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Methods in Molecular Biology 




Second Edith" 



Edited 

Bing-Yuan Ch 
Harry W. Jarred 



^HUMANA PRESS 



METHODS IN MOLECULAR BIOLOGY™ 



John M. Walker, Series Editor 



210. MHC Protocols, edited by Stephen H. Powis and Robert W. 

Vauglmn, 2003 
209. Transgenic Mouse Methods and Protocols, edited by Marten 

Hofker and Jan van Deursen, 2002 
208. Peptide Nucleic Acids: Methods and Protocols, edited by 

Peter E. Nielsen, 2002 
207. Human Antibodies for Cancer Therapy: Reviews and Protocols. 

edited by Martin Welschofandjurgen Krauss, 2002 
206. Endothelin Protocols, edited by Janet J. Maguire and Anthony 

P. Davenport, 2002 
205. E. coli Gene Expression Protocols, edited by Peter E. 

Vaillancourt, 2002 
204. Molecular Cytogenetics: Methods and Protocols, edited by 

Yao-Shan Fan, 2002 
203. In Situ Detection of DNA Damage: Methods and Protocols, 

edited by Vladimir V. Didenko, 2002 
202. Thyroid Hormone Receptors: Methods and Protocols, edited 

by Aria Baniahmad, 2002 
201. Combinatorial Library Methods and Protocols, edited by 

Lisa B.English, 2002 
200. DNA Methylation Protocols, edited by Ken I. Mills and Bemie 

H, Ramsahoye, 2002 
199. Liposome Methods and Protocols, edited by Suhhash C. Basu 

and Manju Basil, 2002 
198. Neural Stem Cells: Methods and Protocols, edited by Tanja 

Zigova, Juan R. Sanchez-Ramos, and Paul R. Sanherg, 2002 
197. Mitochondrial DNA: Methods and Protocols, edited by William 

C. Copeland, 2002 
196. Oxidants and Antioxidants: Ultrastnictural and Molecular 

Biology Protocols, edited by Donald Armstrong, 2002 
195. Quantitative Trait Loci: Methods and Protocols, edited by 

Nicola J. Camp and Angela Cox, 2002 
194. Posttranslational Modifications of Proteins: Toolsfor Func- 
tional Proteomics, edited by Christoph Kannicht, 2002 
193. RT-PCR Protocols, edited by Joseph O'Connell, 2002 
192. PCR Cloning Protocols, 2nd ed., edited by Bing-Yuan Chen 

and Harry W.Janes, 2002 
191. Telomeres and Telomerase: Methods and Protocols, edited 

by John A. Double and Michael J. Thompson, 2002 
190. High Throughput Screening: Methods and Protocols, edited 

by William P. Janzen, 2002 
189. GTPase Protocols: The RAS Superfamily, edited by Edward 

J. Manser and Thomas Leung, 2002 
188. Epithelial Cell Culture Protocols, edited by Clare Wise, 2002 
187. PCR Mutation Detection Protocols, edited by Bimal D. M. 

Theophilus and Ralph Rapley, 2002 
186. Oxidative Stress and Antioxidant Protocols, edited by 

Donald Armstrong, 2002 
185. Embryonic Stem Cells: Methods and Protocols, edited by 

Kursad Turksen, 2002 
184. Biostatistical Methods, edited by Stephen W. Looney, 2002 
183. Green Fluorescent Protein: Applications and Protocols, edited 

by Barry W. Hicks, 2002 
182. In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff 

Braman, 2002 



181. Genomic Imprinting: Methods and Protocols, edited by 

Andrew Ward, 2002 
180. Transgenesis Techniques, 2nd ed.: Principles and Protocols, 

edited by Alan R. Clarke, 2002 
179. Gene Probes: Principles and Protocols, edited by Marilena 

Aquino de Muro and Ralph Rapley, 2002 
178." Antibody Phage Display: Methods and Protocols, edited by 

Philippa M. O'Brien and Robert Aitken, 2001 
177. Two-Hybrid Systems: Methods and Protocols, edited by Paul 

N. MacDonald, 2001 
176. Steroid Receptor Methods: Protocols and Assays, edited by 

Benjamin A. Lieberman, 2001 
175. Genomics Protocols, edited by Michael P. Starkey and 

Ranmath Elaswarapu, 2001 
174. Epstein-Barr Virus Protocols, edited by Joanna B. Wilson 

and Gerhard H.W.May, 2001 
173. Calcium-Binding Protein Protocols, Volume 2: Methods and 

Techniques, edited by Hans J. Vogel, 2001 
172. Calcium-Binding Protein Protocols, Volume 1: Reviews and 

Case Histories, edited by Hans ,1. Vogel, 2001 
171. Proteoglycan Protocols, edited by Renato V. lozzo, 2001 
170. DNA Arrays: Methods and Protocols, edited by Jang B. 

Rampal, 2001 
169. Neurotrophin Protocols, edited by Robert A. Rush, 2001 
168. Protein Structure, Stability, and Folding, edited by Kenneth 

P. Murphy, 2001 
167. DNA Sequencing Protocols, Second Edition, edited by Colin 

A. Graham and Alison J. M. Hill, 2001 
166. Immunotoxin Methods and Protocols, edited by Walter A. Hall, 2001 
165. SV40 Protocols, edited by Leda Raptis, 2001 
164. Kinesin Protocols, edited by lsabelle Vemos, 2001 
163. Capillary Electrophoresis of Nucleic Acids, Volume 2: 

Practical Applications of Capillary Electrophoresis, edited by 

Keith R. Mitchelson and Jing Cheng, 2001 
162. Capillary Electrophoresis of Nucleic Acids, Volume 1: 

Introduction to the Capillary Electrophoresis of Nucleic Acids, 

edited by Keith R. Mitchelson and Jing Cheng, 2001 
161. Cytoskeleton Methods and Protocols, edited by Ray H. Gavin, 2001 
160. Nuclease Methods and Protocols, edited by Catherine H. 

Schein, 2001 
159. Amino Acid Analysis Protocols, edited by Catherine Cooper, 

Nicole Packer, and Keith Williams, 2001 
158. Gene Knockoout Protocols, edited by Martin J. Tvmms and 

Ismail Kola, 2001 
157. Mycotoxin Protocols, edited by Mary W. Trucksess and Albert 

E. Pohland, 2001 
156. Antigen Processing and Presentation Protocols, edited by 

Joyce C. Solheim, 2001 
155. Adipose Tissue Protocols, edited by Gerard Ailhaud, 2000 
154. Connexin Methods and Protocols, edited by Roberto Bruzzone 

and Christian Giaume, 2001 
153. Neuropeptide Y Protocols, edited by Ambikaipakan 

Balasubramaniam, 2000 
152. DNA Repair Protocols: Prokaryotic Systems, edited by Patrick 

Vaughan, 2000 



METHODS IN MOLECULAR BIOLOGY™ 



PCR Cloning 
Protocols 

Second Edition 

Edited by 

Bing-Yuan Chen 

and 

Harry W. Janes 

Rutgers University, 
New Brunswick, NJ 



Humana Press ^i^ Totowa, New Jersey 



© 2002 Humana Press Inc. 
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Cover illustration: The Subtracted cDNA amplification of PBMCs stimulated with PHA. See Fig. 2 on page 107. 

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Printed in the United States of America. 10 987654321 

Library of Congress Cataloging in Publication Data 

Main entry under title: Methods in molecular biology™. 

PCR cloning protocols: second edition / edited by Bing-Yuan Chen and Harry W. Janes.— 2nd ed. 
p. cm. — (Methods in molecular biology ; 192) 
Includes bibliographical references and index. 

ISBN 0-89603-969-2 (hb : alk. paper) - ISBN 0-89603-973-0 (comb. : alk. paper) 
1. Molecular cloning— Laboratory manuals. 2. Polymerase chain reaction—Laboratory 
manuals. I. Chen, Bing-Yuan. II. Janes, Harry W. III. Methods in molecular biology 
(Clifton, N.J.) ; v. 192 

QH442.2 .P37 2002 
572.8'6-dc21 

2001039702 



Preface 



PCR is probably the single most important methodological invention in 
molecular biology to date. Since its conception in the mid-1980s, it has rapidly 
become a routine procedure in every molecular biology laboratory for identify- 
ing and manipulating genetic material, from cloning, sequencing, mutagenesis, 
to diagnostic research and genetic analysis. What's astounding about this inven- 
tion is that new and innovative applications of PCR have been generated with 
stunning regularity; its potential has shown no signs of leveling off. New 
applications for PCR are literally transforming molecular biology. In the post- 
genomic era, PCR has especially become the method of choice to clone existing 
genes and generate a wide array of new genes by mutagenesis and/or recombina- 
tion within the genes of interest. The fast and easy availability of these genes is 
essential for the study of functional genomics, gene expression, protein struc- 
ture-function relationships, protein-protein interactions, protein engineering, and 
molecular evolution. 

PCR Cloning Protocols was prepared in response to the need to have an 
up-to-date compilation of proven protocols for PCR cloning and mutagenesis. It 
builds upon the best-selling first edition, PCR Cloning Protocols: From Molecu- 
lar Cloning to Genetic Engineering, a book in the Methods in Molecular Biol- 
ogy™ series published in 1997. We divided the new edition into five parts. Part 
I. Performing and Optimizing PCR, contains basic PCR methodology, includ- 
ing PCR optimization and computer programs for PCR primer design and analy- 
sis, as well as novel variations for cloning genes of particular characteristics or 
origins, emphasizing long-distance PCR and GC-rich template amplification. 
Part II. Cloning PCR Products, presents both conventional and novel enzyme- 
free and restriction site-free procedures to clone PCR products into various vec- 
tors, either directionally or non-directionally. Part III. Mutagenesis and 
Recombination, addresses the use of PCR to facilitate DNA mutagenesis and 
recombination in various innovative approaches to generate a wide array of 
mutants. Part IV. Cloning Unknown Neighboring DNA, contains a compre- 
hensive collection of protocols to fulfill the frequent and challenging task of 
cloning uncharacterized DNA flanking a known DNA fragment. Finally, Part V. 
Library Construction and Screening, addresses particular applications of PCR 
in library and sublibrary generation and screening. Each part also contains an 
overview, which summarizes the current methods available and their underlying 



vi Preface 

strategies, advantages, and disadvantages for that particular topic. These reviews 
are especially helpful to new researchers to orient themselves with the field and 
to guide them to choose a procedure that is most suitable for their experiments. 

We hope that PCR Cloning Protocols will provide readily reproducible 
laboratory protocols that researchers in the field will follow closely and thereby 
increase their success rate in their experiments. 

We are indebted to Mirah Riben for her superb help during the editing of 
the book. We also thank Prof. John M. Walker, the series editor, for his help, 
advice, and guidance. 

Bing-Yuan Chen 
Harry W. Janes 



Contents 



Preface v 

Contributors xi 

Part I. Performing and Optimizing PCR 

1 Polymerase Chain Reaction: Basic Principles and Routine Practice 
Lori A. Kolmodin and David E. Birch 3 

2 Computer Programs for PCR Primer Design and Analysis 
Bing-Yuan Chen, Harry W. Janes, and Steve Chen 19 

3 Single-Step PCR Optimization 

Using Touchdown and Stepdown PCR Programming 
Kenneth H. Roux 31 

4 XL PCR Amplification of Long Targets from Genomic DNA 

Lori A. Kolmodin 37 

5 Coupled One-Step Reverse Transcription and Polymerase Chain 

Reaction Procedure for Cloning Large cDNA Fragments 
Jyrki T. Aatsinki 53 

6 Long Distance Reverse-Transcription PCR 

Volker Thiel, Jens Herold, and Stuart G. Siddell 59 

7 Increasing PCR Sensitivity for Amplification 

from Paraffin-Embedded Tissues 
Abebe Akalu and Juergen K. V. Reichardt 67 

8 GC-Rich Template Amplification by Inverse PCR: 

DNA Polymerase and Solvent Effects 
Alain Moreau, Da Shen Wang, Steve Forget, Colette Duez, 

and Jean Dusart 75 

9 PCR Procedure for the Isolation of Trinucleotide Repeats 

Teruaki Tozaki 81 

10 Methylation-Specific PCR 

Haruhiko Ohashi 91 



VII 



viii Contents 

1 1 Direct Cloning of Full-Length Cell Differentially Expressed Genes 

by Multiple Rounds of Subtractive Hybridization 
Based on Long-Distance PCR and Magnetic Beads 
Xin Huang, Zhenglong Yuan, and Xuetao Cao 99 

Part II. Cloning PCR Products 

12 Cloning PCR Products: An Overview 

Baotai Guo and Yuping Bi 1 1 1 

13 Using T4 DNA Polymerase to Generate Clonable PCR Products 
KaiWang 121 

14 Enzyme-Free Cloning of PCR Products 

and Fusion Protein Expression 
Brett A. Neilan and Daniel Tillett 125 

15 Directional Restriction Site-Free Insertion of PCR Products 

into Vectors 
Guo Jun Chen 133 

16 Autosticky PCR: 

Directional Cloning of PCR Products with Preformed 5' Overhangs 
Jozsef Gal and Miklos Kalman 141 

17 A Rapid and Simple Procedure for Direct Cloning 

of PCR Products into Baculoviruses 
Tamara S. Gritsun, Michael V. Mikhailov, 
and Ernest A. Gould 153 

Part III. Mutagenesis and Recombination 

18 PCR Approaches to DNA Mutagenesis and Recombination: 

An Overview 
Binzhang Shen 167 

19 In-Frame Cloning of Synthetic Genes Using PCR Inserts 

James C. Pierce 175 

20 Megaprimer PCR 

Sailen Barik 189 

21 PCR-Mediated Recombination: 

A General Method Applied to Construct Chimeric Infectious 
Molecular Clones 
Guowei Fang, Barbara Weiser, Aloise Visosky, Timothy Moran, 
and Harold Burger 197 

22 PCR Method for Generating Multiple Mutations at Adjacent Sites 

Jiri Adamec 207 



Contents ix 

23 A Fast Polymerase Chain Reaction-Mediated Strategy for Introducing 

Repeat Expansions into CAG-Repeat Containing Genes 
Franco Laccone 277 

24 PCR Screening in Signature-Tagged Mutagenesis of Essential Genes 
Dario E. Lehoux and Roger C. Levesque 225 

25 Staggered Extension Process (StEP) In Vitro Recombination 

Anna Marie Aguinaldo and Frances Arnold 235 

26 Random Mutagenesis by Whole-Plasmid PCR Amplification 
Donghak Kim and F. Peter Guengerich 241 

Part IV. Cloning Unknown Neighboring DNA 

27 PCn-Based Strategies to Clone Unknown DNA Regions 

from Known Foreign Integrants: An Overview 
Eric Ka-Wai Hui, Po-Ching Wang, and Szecheng J. Lo 249 

28 Long Distance Vectorette PCR (LDV PCR) 

James A. L. Fenton, Guy Pratt, and Gareth J. Morgan 275 

29 Nonspecific, Nested Suppression PCR Method 

for Isolation of Unknown Flanking DNA ("Cold-Start Method") 
Michael Lardelli 285 

30 Inverse PCR: cDNA Cloning 

Sheng-He Huang 293 

31 Inverse PCR: Genomic DNA Cloning 
Ambrose Y. Jong, Anna Tang, De-Pei Liu, 

and Sheng-He Huang 301 

32 Gene Cloning and Expression Profiling by Rapid Amplification 

of Gene Inserts with Universal Vector Primers 
Sheng-He Huang, Hua-Yang Wu, and Ambrose Y. Jong 309 

33 The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions 

by Inverse PCR 
Vincent J. J. Martin and William W. Mohn 315 

34 Rapid Amplification of Genomic DNA Sequences Tagged 

by Insertional Mutagenesis 
Martina Celerin and Kristin T. Chun 325 

35 Isolation of Large Terminal Sequences of BAC Inserts Based 

on Double-Restriction-Enzyme Digestion Followed 
by Anchored PCR 
Zhong-Nan Yang and T. Erik Mirkov 337 



x Contents 

36 A "Step Down" PCR-Based Technique for Walking 

Into and the Subsequent Direct Sequence Analysis 
of Flanking Genomic DNA 
Ziguo Zhang and Sarah Jane Gun 343 

Part V. Library Construction and Screening 

37 Use of PCR in Library Screening: An Overview 

JinbaoZhu 353 

38 Cloning of Homologous Genes by Gene-Capture PCR 

Renato Mastrangeli and Silvia Donini 359 

39 Rapid and Nonradioactive Screening of Recombinant Libraries by PCR 
Michael W. King 377 

40 Rapid cDNA Cloning by PCR Screening (RC-PCR) 

Toru Takumi 385 

41 Generation and PCR Screening of Bacteriophage A Sublibraries 
Enriched for Rare Clones (the "Sublibrary Method") 

Michael Lardelli 391 

42 PCR-Based Screening for Bacterial Artificial Chromosome Libraries 
Yuji Yasukochi 401 

43 A 384-Well Microtiter-Plate-Based Template Preparation 

and Sequencing Method 
Lei He and Kai Wang 4 1 1 

44 A Microtiter-Plate-Based High Throughput PCR Product 

Purification Method 

Ryan Smith and Kai Wang 417 

Index 423 



Contributors 



Jyrki T. Aatsinki • Institute of Dentistry, University ofOulu, Finland 

Jiri Adamec • Mayo Clinic and Foundation, Rochester, MN 

Anna Marie Aguinaldo • Division of Chemistry and Chemical Engineering, 

California Institute of Technology, Pasadena, CA 
Abebe Akalu • Institute for Genetic Medicine, USC School of Medicine, 

Los Angeles, CA 
Frances Arnold • Division of Chemistry and Chemical Engineering, 

California Institute of Technology, Pasadena, CA 
Sailen Barik • Department of Biochemistry and Molecular Biology, University 

of South Alabama, Mobile, AL 
Yuping Bi • Institute of Plant Biotechnology, Shangdong Academy 

of Agricultural Sciences, Jinan, China 
Da id E. Birch • Roche Molecular Systems, Alameda, CA 
Harold Burger • Wadsworth Center, Albany, NY 
Xuetao Cao • Department of Immunology, Second Military Medical 

University, Shanghai, China 
Martina Celerin • Department of Biology, Indiana University, Bloomington, IN 
Bing-Yuan Chen • Department of Plant Science, Rutgers University, New 

Brunswick, NJ 
Guo Jun Chen • F. Hoffmann La-Roche, Basel, Switzerland 
Steve Chen • NetOsprey Inc., Berkeley, CA 
Kristin T. Chun • Department of Pediatrics, Indiana University School 

of Medicine, Indianapolis, IN 
Silvia Donini • Istituto di Ricerca Cesare Serono, Rome, Italy 
Colette Duez • Centre D'Ingenierie des Proteines, Universite de Liege, 

Liege, Belgium 
Jean Dusart • Centre D'Ingenierie des Proteines, Universite de Liege, 

Liege, Belgium 
Guowei Fang • Wadsworth Center, Albany, NY 
James A. L. Fenton • Department of Molecular Oncology, University of Leeds, 

Leeds, UK 
Steve Forget • Sainte-Justine Hospital Research Center, Montreal, Canada 
Jozsef Gal • Institute for Biotechnology, Bay Zoltdn Foundation for Applied 

Research, Szeged, Hungary 

xi 



xii Contributors 

Ernest A. Gould • CEH Oxford, Oxford, UK 

Tamara S. Gritsun • CEH Oxford, Oxford, UK 

F. Peter Guengerich • Department of Biochemistry and Center in Molecular 

Toxicology, Vanderbilt University School of Medicine, Nashville, TN 
Baotai Guo • Institute of Plant Biotechnology, Laiyang Agricultural College, 

Shandong, China 
Sarah Jane Gurr • Department of Plant Sciences, University of Oxford, 

Oxford, UK 
Lei He • PhenoGenomics Corp., Bothell, WA 
Jens Herold • SWITCH -Biotech AG, Martinsried, Germany 
Sheng-He Huang • Department of Pediatrics, University of Southern California, 

Los Angeles, CA 
Xin Huang • Department of Immunology, Second Military Medical University, 

Shanghai, China 
Eric Ka-Wai Hui • Department of Microbiology, Immunology and Molecular 

Genetics, University of California Los Angeles, Los Angeles, CA 
Harry W. Janes • Department of Plant Science, Rutgers University, New 

Brunswick, NJ 
Ambrose Y. Jong • Department of Pediatrics, University of Southern California, 

Los Angeles, CA 
MiklOs Kalman • Institute for Biotechnology, Bay Zoltdn Foundation 

for Applied Research, Szeged, Hungary 
Donghak Kim • Department of Biochemistry and Center in Molecular 

Toxicology, Vanderbilt University School of Medicine, Nashville, TN 
Michael W. King • Department of Biochemistry and Molecular Biology, 

Indiana University School of Medicine, Terre Haute, IN 
Lori A. Kolmodin • Roche Molecular Systems, Pleasanton, CA 
Franco Laccone • Institute of Human Genetics, University ofGoettingen, 

Goettingen, Germany 
Michael Lardelli • Department of Molecular Biosciences, Adelaide 

University, Australia 
Dario E. Lehoux • Health and Life Sciences Research Center, Universite 

Laval, Sainte-Foy, Quebec, Canada 
Roger C. Levesque • Health and Life Sciences Research Center, Universite 

Laval, Sainte-Foy, Quebec, Canada 
De-Pei Liu • Chinese Academy of Medical Sciences and Peking Union Medical 

College, Beijing, China 
Szecheng J. Lo • Institute of Microbiology and Immunology, National Yang-Ming 

University, Taipei, Taiwan, ROC 



Contributors xiii 

Vincent J. J. Martin • Department of Chemical Engineering, University 

of California, Berkeley, CA 
Renato Mastrangeli • Istituto di Ricerca Cesare Serono, Rome, Italy 
Michael V. Mikhailov • CEH Oxford, Oxford, UK 
T. Erik Mirkov • Department of Plant Pathology and Microbiology, 

The Texas A&M University Agricultural Experiment Station, Weslaco, TX 
William W. Mohn • Department of Microbiology and Immunology, 

University of British Columbia, Vancouver, Canada 
Timothy Moran • Wadsworth Center, Albany, NY 

Alain Moreau • Sainte-Justine Hospital Research Center, Montreal, Canada 
Gareth J. Morgan • Department of Molecular Oncology, University of Leeds, 

Leeds, UK 
Brett A. Neilan • School of Microbiology and Immunology, the University 

of New South Wales, Sydney, Australia 
Haruhiko Ohashi • Nagoya National Hospital, Nagoya, Japan 
James C. Pierce • University of the Sciences in Philadelphia, Philadelphia, PA 
Guy Pratt • Department of Molecular Oncology, University of Leeds, Leeds, UK 
Juergen K.V. Reichardt • Institute for Genetic Medicine, USC School 

of Medicine, Los Angeles, CA 
Kenneth H. Roux • Department of Biological Science, Florida State University, 

Tallahassee, FL 
Binzhang Shen • Department of Molecular Biology, Massachusetts General 

Hospital, Boston, MA 
Stuart G. Siddell • Institute of Virology and Immunology, University 

ofWurzburg, Wiirzburg, Germany 
Ryan Smith • PhenoGenomics Corp., Bothell, WA 
Anna T'ang • Department of Pathology, University of Southern California, 

Los Angeles, CA 
Toru Takumi • Osaka Bioscience Institute, Osaka, Japan 
Volker Thiel • Institute of Virology and Immunology, University ofWurzburg, 

Wiirzburg, Germany 
Daniel Tillett • School of Microbiology and Immunology, University 

of New South Wales, Sydney, Australia 
Teruaki Tozaki • Department of Molecular Genetics, Laboratory of Racing 

Chemistry, Utsunomiya, Tochigi, Japan 
Aloise Visosky • Wadsworth Center, Albany, NY 

Da Shen Wang • Sainte-Justine Hospital Research Center, Montreal, Canada 
Kai Wang • PhenoGenomics Corp., Bothell, WA 
Po-Ching Wang • Department of Medicine, National Yang-Ming University, 

Taipei, Taiwan, ROC 



xiv Contributors 

Barbara Weiser • Wadsworth Center, Albany, NY 

Hua-Yang Wu • Department ofPediatricsx, University of Southern California, 

Los Angeles, CA 
Zhong-Nan Yang • Department of Plant Pathology and Microbiology, 

The Texas A&M University Agricultural Experiment Station, Weslaco, TX 
Yuji Yasukochi • National Institute of Agrobiological Sciences, Ibaraki, Japan 
Zhenglong Yuan • Department of Immunology, Second Military Medical 

University, Shanghai, China 
Ziguo Zhang • Department of Plant Sciences, University of Oxford, Oxford, UK 
Jinbao Zhu • Department of Genetics and Plant Breeding, China Agricultural 

University, Beijing, China 



Performing and Optimizing PCR 



Polymerase Chain Reaction 

Basic Principles and Routine Practice 
Lori A. Kolmodin and David E. Birch 

1. Introduction 

1 . 1. PCR Definition 

The polymerase chain reaction (PCR) is a primer-mediated enzymatic amplifica- 
tion of specifically cloned or genomic DNA sequences (1). This PCR process, invented 
more than a decade ago, has been automated for routine use in laboratories worldwide. 
The template DNA contains the target sequence, which may be tens or tens of thou- 
sands of nucleotides in length. A thermostable DNA polymerase such as Taq DNA 
polymerse, catalyzes the buffered reaction in which an excess of an oligonucleotide 
primer pair and four deoxynucleoside triphosphates (dNTPs) are used to make mil- 
lions of copies of the target sequence. Although the purpose of the PCR process is to 
amplify template DNA, a reverse transcription step allows the starting point to be 
RNA (2-5). 

1.2. Scope of PCR Applications 

PCR is widely used in molecular biology and genetic disease studies to identify 
new genes. Viral targets, such as HIV-1 and HCV, can be identified and quantified by 
PCR. Active gene products can be accurately quantitated using RNA-PCR. In such 
fields as anthropology and evolution, sequences of degraded ancient DNAs can be 
tracked after PCR amplification. With its exquisite sensitivity and high selectivity, 
PCR has been used in wartime human identification and validation in crime labs for 
mixed-sample forensic casework. In the realm of plant and animal breeding, PCR tech- 
niques are used to screen for traits and to evaluate living four-cell embryos. Environ- 
mental and food pathogens can be quickly identified and quantitated at high sensitivity 
in complex matrices with simple sample preparation techniques. 



From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 



4 Kolmodin and Birch 

1.3. PCR Process (see Note 1) 

The PCR process requires a repetitive series of the three fundamental steps that 
defines one PCR cycle: double-stranded DNA template denaturation, annealing of two 
oligonucleotide primers to the single-stranded template, and enzymatic extension of 
the primers to produce copies that can serve as templates in subsequent cycles. The 
target copies are double-stranded and bounded by annealing sites of the incorporated 
primers. The 3' end of the primer should complement the target exactly, but the 5' end 
can actually be a noncomplementary tail with restriction enzyme and promotor sites 
that will also be incorporated. As the cycles proceed, both the original template and 
the amplified targets serve as substrates for the denaturation, primer annealing, and 
primer extension processes. Since every cycle theoretically doubles the amount of 
target copies, a geometric amplification occurs. Given an efficiency factor for each 
cycle, the amount of amplified target Y produced from an input copy number X after n 
cycles is 

Y = X(l = efficiency) 11 (1) 

With this amplification power, 25 cycles could produce 33 million copies. Every 
extra 10 cycles produces 1024 more copies. Unfortunately, the process becomes self- 
limiting and amplification factors are generally between 10 5 - and 10 9 -fold. Excess 
primers and dNTPs help drive the reaction that commonly occurs in 10 mM Tris-HCl 
buffer, pH 8.3 (at room temperature). In addition, 50 mM KC1 is present to provide 
proper ionic strength and magnesium ion is required as an enzyme cof actor (6). 

The denaturation step occurs rapidly at 94-96 c C. Primer annealing depends on the 
T m , or melting temperature, of the primer:template hybrids. Generally, one uses a pre- 
dictive software program to compute the T m s based on the primer's sequence, their 
matched concentrations, and the overall salt concentration. The best annealing tem- 
perature is determined by optimization. Extension occurs at 72°C for most templates. 
PCR can also easily occur with a two-temperature cycle consisting of denaturation and 
annealing/extension. 

1.4. Carryover Prevention 

PCR has the potential sensitivity to amplify single molecules, so PCR products that 
can serve as templates for subsequent reactions must be kept isolated after amplifica- 
tion. Even tiny aerosols can contain thousand of copies of carried-over target mol- 
ecules that can convert a true negative into a false positive. In general, dedicated 
pipetors, pipet tips with filters, and separate work areas should be considered and/or 
designated for RNA or DNA sample preparation, reaction mixture assemblage, the 
PCR process, and the reaction product analysis. As with any high sensitivity tech- 
nique, the judicious and frequent use of positive and negative controls is required for 
each amplification (7-9). Through the use of dUTP instead of dTTP for all PCR 
samples, it is possible to design an internal biochemical mechanism to attack the PCR 
carryover problem. These PCR products are dU-containing and can be cloned, 
sequenced, and analyzed as usual. Pretreatment of each PCR reaction with uracil-N 
glycosylase (UNG), which catalyzes the removal of uracil from single- and double- 



PCR: Basic Principles 5 

stranded DNA, will destroy any PCR product carried over from previous reactions, 
leaving the native T-containing sample ready for amplification (10). 

1.5. Hot Start 

PCR is conceptualized as a process that begins when thermal cycling ensues. The 
annealing temperature sets the specificity of the reaction, assuring that the primary 
primer binding events are the ones specific for the target in question. In preparing a 
PCR amplification on ice or at room temperature, however, the reactants are all present 
for nonspecific primer annealing to any single- stranded DNA present. Because DNA 
polymerases have some residual activity even at lower temperatures, it is possible to 
extend these misprimed hybrids and begin the PCR process at the wrong sites. To 
prevent this mispriming/misextension, a number of "Hot Start" strategies have been 
developed. In Hot Start PCR, a key reaction component essential for polymerase 
activity is withheld or separated from the reaction mixture until an elevated tempera- 
ture is reached (11,12). 

To separate an essential component from the reaction mixture in order to delay 
amplification, the following techniques can be utilized: 

1.5.1. Manual Hot Start 

In Manual Hot Start, a key reaction component such as Taq DNA polymerase or 
MgCl 2 is withheld from the original amplification mixture and added to the reaction 
when the temperature within the tube exceeds the optimal annealing temperature, i.e., 
above 65°-70°C. 

1.5.2. Physical Barrier Hot Start, i.e., AmpliWax® PCR Gems 
from Applied Biosystems 

In AmpliWax PCR gem-facilitated Hot Start, reaction components are divided into 
two mixes, and separated by a solid wax layer within the reaction tube (11). During the 
initial denaturation step, the wax layer melts at 75°-80°C allowing the two reaction 
mixes to combine through thermal convection. 

1.5.3. Monoclonol Antibodies to DNA Polymerases Hot Start, 
i.e., PfuTurbo® Hotstart DNA polymerase from Stratagene 
orJaqStart from Clontech 

In polymerase-antibody Hot Start, a PCR preincubation step is added, during which 
a heat-sensitive antibody attaches to the DNA polymerase [Taq or recombinant 
Thermus thermophilics (rTth)] inactiving the enzyme within the reaction mixture. As 
the temperature within the tubes rises, the antibody detaches and is inactivated, setting 
the polymerase free to begin polymerization. 

1.5.4. Modified DNA Polymerases for Hot Start, i.e., AmpliTaq Gold® 
from Applied Biosytems 

With AmpliTaq Gold, Hot Start is achieved with a chemically modified Taq DNA 
polymerase. The modification blocks the polymerase activity until it is reversed by a 
high temperature, pre-PCR incubation (e.g., 95°C for >10 min). The pre-PCR incuba- 



6 Kolmodin and Birch 

tion links directly to the denaturation step of the first PCR cycle. So, the reaction 
mixture never sees active polymerase below the optimal primer annealing tempera- 
ture. If the pre-PCR incubation is omitted, the modification is reversed during the PCR 
cycling, and polymerase activity increases slowly. In addition to a Hot Start, this pro- 
vides a time release effect, where polymerase activity builds as the DNA substrate 
accumulates (12). 

1.5.5. Oligonucleotide Inhibitors of DNA Polymerases for Hot Start 

In polymerase-inhibitor Hot Start, DNA polymerase-binding oligonucleotides are 
added to the PCR amplification, keeping the enzyme inactive at ambient temperatures. 
Increasing the temperature dissociates the inhibitor from the enzyme, setting it free to 
begin polymerization. Moreover, inhibition is thermally reversible (13-16). 

1.6. PCR Achievements 

PCR has been used to speed the human genome discovery and for early detection of 
viral diseases. Single sperm cells to measure crossover frequencies can be analyzed 
and four-cell cow embryos can be typed. Trace forensic evidence of even mixed 
samples can be analyzed. Single-copy amplification requires some care, but is feasible 
for both DNA and RNA. True needles in haystacks can be found simply by amplifying 
the needles. PCR facilitates cloning of DNA sequences and forms a natural basis for 
cycle sequencing by the Sanger method (17). In addition to generating large amounts 
of template for cycle sequencing, PCR has been used to map chromosomes and to 
analyze both large and small changes in chromosome structure. 

1.7. PCR Enzymes 

The choice of the DNA polymerase is determined by the aims of the experiment. 
There are a variety of commercially available enzymes to choose from that differ in 
their thermal stability, processivity, and fidelity as depicted in Table 1. The most com- 
monly used and most extensively studied enzyme is Taq DNA polymerase, e.g., 
AmpliTaq® DNA polymerase. 

1.7.1. AmpliTaq DNA Polymerase 

AmpliTaq DNA Polymerase (Applied Biosystems, Foster City, CA) is a highly 
characterized recombinant enzyme for PCR. It is produced in Escherichia coli (E. 
coll) from the Taq DNA polymerase gene, thereby assuring high purity. It is com- 
monly supplied and used as a 5 U/jiL solution in buffered 50% (v/v) glycerol (18). 

1. Biophysical Properties. The enzyme is a 94-kDa protein with a 5'-3' polymerization ac- 
tivity that is most efficient in the 70°-80°C range. This enzyme is very thermostable, with 
a half-life at 95°C of 35-40 min. In terms of thermal cycling, the half-life is approx 100 
cycles. PCR products amplified using AmpliTaq DNA polymerase will often have single 
base overhangs on the 3' ends of each polymerized strand, and this artifact can be suc- 
cessfully exploited for use with T/A cloning vectors. 

2. Biochemical Reactions. DNA Polymerase requires magnesium ion as a cofactor and cata- 
lyzes the extension reaction of a primed template at 72°C. The four dNTPs (consisting of 



PCR: Basic Principles 



7 



Table 1 

Some Commercially Available DNA Polymerases and Associated Properties (18) 











Exonuclease 




DNA 




Commercial 


95°C 


Activity 


Extension Rate 


Polymerase 


Source 


Name 


Half-life 


5-3' 3-5' 


(nucleosides/s) 


Taq 


Thermus 
aquaticus 


AmpliTaq 


40min 


+ 


75 


Pwo 


Pyrococcus 
woesei 




? 


+ 


9 


Pfu 


Pyrococcus 
furiosus 




>120 min 


+ 


60 


vTth 


Thermus 

thermophilus 




20 min 


+ 


60 


Tfl 


Thermas flavus 




? 


- 


? 


Tli 


Thermus litoris 


Vent 


400 min 


+ 


67 


Tma 


Thermotoga 
maritima 




>50 min 


+ 


? 



dATP, dCTP, dGTP, and dTTP or dUTP) are used according to the basepairing rule to 
extend the primer and thereby to copy the target sequence. Modified nucleotides (ddNTPs, 
biotin-11-dNTP, dUTP, deaza-dGTP, and flourescently labeled dNTPs) can be incorpo- 
rated into PCR products. 
3. Associated Activities. AmpliTaq DNA Polymersae has a fork-like structure-dependent, 
polymerization enhanced, 5—3' nuclease activity. This activity allows the polymerase to 
degrade downstream primers and indicates that circular targets should be linearized before 
amplification. In addition, this nuclease activity has been employed in a fluorescent sig- 
nal-generating technique for PCR quantitation (19). AmpliTaq DNA Polymersae does 
not have an inherent 3'-5' exonuclease or proofreading activity, but produces amplicons 
of sufficient high fidelity for most applications. 

1.7.2. AmpliTaq Gold 

AmpliTaq Gold (Applied Biosystems, Foster City, CA) is chemically modified 
AmpliTaq DNA polymerase. The reversible modification keeps the enzyme inactive 
at room temperature. High temperature and low pH promote the reversal, restoring the 
enzyme activity. These conditions occur in a Tris-buffered PCR at 92°-95°C (Tris-Cl 
formulated to pH 8.3 at 25°C drops below pH 7.0 above 90°C). AmpliTaq Gold is 
formulated to perform the same as 5 U/|J,L AmpliTaq DNA polymerase. Therefore, a 
hot start can be added to most PCRs optimized with AmpliTaq DNA polymerase by 
substituting AmpliTaq Gold and adding a 10-min, 95 C C, pre-PCR, activation step. 
The same results can be achieved without the pre-PCR activation step by adding an 
additional 10 or more PCR cycles. Under these conditions, the enzyme is activated 
incrementally during the PCR denaturation steps. 



8 Kolmodin and Birch 

1.8. Primers 

PCR Primers are short oligodeoxyribonucleotides, or oligomers, that are designed to 
complement the end sequences of the PCR target amplicon. These synthetic DNAs are 
usually 15-25 nucleotides long and have approx 50-60% G + C content. Because each 
of the two PCR primers is complementary to a different individual strand of the target 
sequence duplex, the primer sequences are not related to each other. In fact, special care 
must be taken to assure that the primer sequences do not form duplex structures with 
each other or hairpin loops within themselves. The 3' end of the primer must match the 
target in order for polymerization to be efficient, and allele-specific PCR strategies take 
advantage of this fact. In screening for potential sequences and their homology, primer 
design software packages such as Oligo® (National Biosciences, Plymouth, NC) and 
online search sites such as BLAST (NCBI, www.ncbi.nlm.nih.gov/BLAST/), can be 
utilized. To screen for mutants, a primer complementary to the mutant sequence is used 
and results in PCR positives, whereas the same primer will be a mismatch for the 
wild type and does not amplify. The 5' end of the primer may have sequences that 
are not complementary to the target and that may contain restriction sites or promo- 
tor sites that are also incorporated into the PCR product. Primers with degenerate 
nucleotide positions every third base may be synthesized in order to allow for ampli- 
fication of targets where only the amino acid sequence is known. In this case, early 
PCR cycles are peformed with low, less stringent annealing temperatures, followed 
by later cycles with high, more stringent annealing temperatures. 

A PCR primer can also be a homopolymer, such as oligo (dT) 16 , which is often used 
to prime the RNA PCR process. In a technique called RAPDS (randomly amplified 
polymorphic DNAs), single primers as short as decamers with random sequences are 
used to prime on both strands, producing a diverse array of PCR products that form a 
fingerprint of a genome (20). Often, logically designed primers are less successful in 
PCR than expected, and it is usually advisable to try optimization techniques for a 
practical period of time before trying new primers frequently designed near the origi- 
nal sites. 

1.8.1. T m Predictions 

DNA duplexes, such as primer-template complexes, have a stability that depends 
on the sequence of the duplex, the concentrations of the two components, and the salt 
concentration of the buffer. Heat can be used to disrupt this duplex. The temperature at 
which half the molecules are single-stranded and half are double-stranded is called the 
T m of the complex. Because of the greater number of intermolecular hydrogen bonds, 
higher G+C content DNA has a higher T m than lower G+C content DNA. Often, G + C 
content alone is used to predict the T m of the DNA duplex, however, DNA duplexes 
with the same G + C content may have different T m values. A simple, generic formula 
for calculating the T m is: T m = 4(G+C) + 2(A+T) °C. A variety of software packages 
are available to perform more accurate T m predictions using sequence information 
(nearest neighbor analysis) and to assure optimal primer design, e.g., Oligo, BLAST, 
or Melt (Mt. Sinai School of Medicine, New York, NY). 



PCR: Basic Principles 9 

Because the specificity of the PCR process depends on successful primer binding 
events at each amplicon end, the annealing temperature is selected based on the con- 
sensus of the melting temperatures (within 2- 4°C) of the two primers. Usually, the 
annealing temperature is chosen a few degrees below the consensus annealing tem- 
peratures of the primers (1). Different strategies are possible, but lower annealing tem- 
peratures should be tried first to assess the success of amplification to find the 
stringency required for best product specificity. 

1.9. PCR Samples 

1.9.1. Types 

The PCR sample type may be single- or double-stranded DNA of any origin — 
animal, bacterial, plant, or viral. RNA molecules, including total RNA, poly (A+) 
RNA, viral RNA, tRNA, or rRNA, can serve as templates for amplification after con- 
version to so-called complementary DNA (cDNA) by the enzyme reverse transcriptase 
(either MuLV or recombinant, rTth DNA polymerase) (21,22). 

1.9.2. Amount 

The amount of starting material required for PCR can be as little as a single mol- 
ecule, compared to the millions of molecules needed for standard cloning or molecular 
biological analysis. As a basis, up to nanogram amounts of DNA cloned template, up 
to microgram amounts of genomic DNA, or up to 10 5 DNA target molecules are best 
for initial PCR testing. 

1.9.3. Purity 

Overall, the purity of the DNA sample to be subjected to PCR amplification need 
not be high. A single cell, a crude cell lysate, or even a small sample of degraded DNA 
template is usually adequate for successful amplification. The fundamental require- 
ments of sample purity must be that the target contains at least one intact DNA strand 
encompassing the amplified region and that the impurities associated with the target 
be adequately dilute so as to not inhibit enzyme activity. However, for some amplifi- 
cations, such as long PCR, it may be necessary to consider the quality and quantity of 
the DNA sample (23,24). For example, 

1. When more template molecules are available, there is less occurrences of false positives 
caused by either cross-contamination between samples or "carryover" contamination from 
previous PCR amplifications; 

2. When the PCR amplifications lacks specificity or efficiency, or when the target sequences 
are limited, there is a greater chance of inadequate product yield; and 

3. When the fraction of starting DNA available to PCR is uncertain, it is increasingly diffi- 
cult to determine the target DNA content (25). 

1. 10. Other Parameters for Successful PCR 

1.10.1. Metal Ion Cof actors 

Magnesium chloride is an essential cofactor for the DNA polymerase used in PCR, 
and its concentration must be optimized for every primentemplate system. Many com- 



10 Ko I mod in and Birch 

ponents of the reaction bind magnesium ion, including primers, template, PCR prod- 
ucts and dNTPs. The main 1 : 1 binding agent for magnesium ion is the high concentra- 
tion of dNTPs in the reaction. Because it is necessary for free magnesium ion to serve 
as an enzyme cofactor in PCR, the total magnesium ion concentration must exceed the 
total dNTP concentration. Typically, to start the optimization process, 1 .5 mM magne- 
sium chloride is added to PCR in the presence of 0.8 mM total dNTPs. This leaves 
about 0.7 mM free magnesium for the DNA polymerase. In general, magnesium ion 
should be varied in a concentration series from 1.5-4.0 mM in 0.5 mM steps (1,25). 

1. 10.2. Substrates and Substrate Analogs 

DNA polymerases incorporate dNTPs very efficiently, but can also incorporate 
modified substrates, when they are used as supplemental components in PCR. 
Digoxigenin-dUTP, biotin-1 1-dUTP, dUTP, c7deaza-dGTP, and fluorescently labeled 
dNTPs all serve as substrates for DNA polymerases. For conventional PCR, the con- 
centration of dNTPs remains balanced in equimolar ratios, e.g., 200 u,M each dNTP 
(1). However, deviations (from these standard recommendations) may be beneficial in 
certain amplications. For example, when random mutagenesis of a specific target is 
desired, unbalanced dNTP concentrations promote a higher degree of misincorpora- 
tions by the DNA polymerase. 

1.10.3. Buffers and Salts 

The optimal PCR buffer concentration, salt concentration, and pH depend on the 
DNA polymerase in use. The PCR buffer for Taq DNA polymerase consists of 50 mM 
KC1 and 10 mM Tris-HCl, pH 8.3, at room temperature. This buffer provides the ionic 
strength and buffering capacity needed during the reaction. It is important to note that 
the salt concentration affects the T m of the primer: template duplex, and hence the 
annealing temperature. 

1.10.4. Cosolvents 

A variety of PCR cosolvents have been utilized to increase the yield, efficacy, and 
specificity of PCR amplifications. Although these cosolvents are advantageous in some 
amplifications, it is impossible to predict which additive will be useful for each 
primentemplate duplex and therefore the cosolvent must be empirically tested for 
each combination. Some of the more popular cosovents currently in use are listed in 
Table 2 along with the recommended testing ranges (26). 

1.10.5. Thermal Cycling Considerations 
1.10.5.1. PCR Vessels 

PCR must be performed in vessels that are compatible with low amounts of enzyme 
and nucleic acids and that have good thermal transfer characteristics. Typically, 
polypropylene is used for PCR vessels and conventional, thick-walled microcentrifuge 
tubes are chosen for many thermal cycler systems. PCR is most often performed at a 
10-100 u,L reaction scale and requires the prevention of the evaporation/condensation 
processes in the closed reaction tube during thermal cycling. A mineral oil overlay or 



PCR: Basic Principles 



11 



Table 2 

PCR Cosolvents 



Cosolvent 



Recommended Testing 
Ranges 



Comments 



Betaine 



Bovine serum albumin 
(BSA) 

7-deaza-2'- 

deoxyguanosine 

(dC 7 GTP) 

DMSO 



Formamide 



Glycerol 



T4 gene 32 protein 



Final concentration: 
1.0-1.7 M 

10-100 [ig/mL 



Ratio 3:1 
dC 7 GTP:dGTP 



2-10% 



1-5% 



1-10% 



Nonionic detergents: 0.1-1% 

Triton X-100, Tween 20 

NP40 



20-150 [xg/mL 



Tetramehylammonium Final concentration: 

chloride (TMAC) 15-100 mM 



TMA oxalate 



2mM 



Reduces the formation of 
secondary structure caused by 
GC-rich regions (27) 

A nonspecific enzyme stabilizer 
which also binds certain DNA 
inhibitors (28) 

Facilitates amplification of 
templates with stable secondary 
structures when used in place of 
dGTP (1) 

10% reduces Taq activity by 50%. 
Thought to reduce secondary 
structure. Useful for GC rich 
templates. Presumed to lower the 
Tm of the target nucleic acids. 

Improve the specificity of PCR at 
lower denaturation temperatures 
(21,29) 

Improves the thermal stability of 
DNA Polymerases. Improves the 
amplification of high GC templates 
(30) 

Stabilizes Taq DNA polymerase. 
May suppress the formulation of 
secondary structure. May increase 
yield but may also increase non- 
specific amplification. 

Enhance PCR product yield and 
relieve inhibition (31) 

To eliminate nonspecific priming. 
Also used to reduce potential DNA- 
RNA mismatch. Improves the strin- 
gency of hybridization reactions. 

Decreases the formation of nonspe- 
cific DNA fragments and increases 
PCR product yield (32) 



12 Kolmodin and Birch 

wax layer serves this purpose. More recently, 0.2-mL thin-walled vessels have been 
optimized for the PCR process and oil-free thermal cyclers have been designed that 
use a heated cover over the tubes held within the sample block. 

1 .1 0.5.2. Temperature and Time Optimization 

It is essential that the reaction mixtures reach the denaturation, annealing, and exten- 
sion temperatures in each thermal cycle. If insufficient hold time is specified at any 
temperature, the temperature of the sample will not be equilibrated with that of the 
sample block. Some thermal cycler designs time the hold interval based on the block 
temperature, whereas others base the hold time on predicted sample temperature. 

If a conventional thick-walled tube used in a cycler controlled by block tempera- 
ture, a 60-s hold time is sufficient for equilibration. Extra time may be recommended 
at the (72°C) extension step for longer PCR products (23). Using a thin- walled 
0.2-mL tube in a cycler controlled by predicted sample temperature, only 15 s is 
required. To use existing protocols or to development protocols for use at multiple 
laboratories, it is very important to choose hold times according to the cycler design 
and tube wall thickness. 

1.10.6. PCR Amplification Cycles 

The number of PCR amplification cycles should be optimized with respect to the 
starting concentration of the target DNA. Innis and Gelfand (7) recommend from 40- 
45 cycles to amplify 50 target molecules, and 25-30 cycles to amplify 3 x 10 5 mol- 
ecules to the same concentration. This nonproportionality is caused by a so-called 
plateau effect, in which a decrease in the exponential rate of product accumulation 
occurs in late stages of a PCR. This may be caused by degradation of reactants (dNTPs, 
enzyme); reactant depletion (primers, dNTPs); end-product inhibition (pyrophosphate 
formation); competition for reactants by non-specific products; or competition for 
primer binding by reannealing of concentrated (10 nM) product. It is usually advisable to 
run the minimum number of cycles needed to see the desired specific product, because 
unwanted nonspecific products will interfere if the number of cycles is excessive. 

1.10.7. Enzyme/Target 

In a standard aliquot of Taq DNA polymerase used for a 100-u.L reaction, there 
are about 10 10 molecules. Each PCR sample should be evaluated for the number of 
target copies it contains or may contain. For example, 1 ng of lambda DNA contains 
1.8 x 10 7 copies. For low-input copy number PCR, the enzyme becomes limiting and 
it may be necessary to give the extension process incrementally more time. Thermal 
cyclers can reliably perform this automatic segment extension procedure in order to 
maximize PCR yield (1,25). 

1.10.8. Hot Start 

All of the above optimizations also apply to a PCR that is designed, from the begin- 
ning, with a hot start method. Often, a hot start can be incorporated successfully into a 



PCR: Basic Principles 13 

previously optimized PCR without changing the reaction conditions. However, it usu- 
ally pays to reoptimize after adding a hot start. Optimization is often a balance between 
producing as much product as possible and overproducing nonspecific, background 
amplifications. Because hot start greatly reduces background amplifications, the upper 
restraints are raised on conditions such as enzyme concentration, cycle number, and 
metal ion cofactor concentration. Sensitive PCRs that have been highly tuned without 
a hot start may fail when a hot start is added. This can be caused by slight delays in 
early cycles caused by mixing or enzyme activation. The PCR usually can be restored, 
often with substantial increase in specific product, by detuning — that is, simply 
increasing limiting parameters or reagents. In addition, there are optimizations spe- 
cific to each hot start method. Mixing or enzyme activation can be affected by PCR 
volume, buffer composition and pH, cosolvents, cycling conditions, and so on. The 
specific product's literature, often a product insert, should be consulted for informa- 
tion on these considerations. 

2. Materials 

The protocol described later illustrates the basic principles and techniques of PCR 
and can be modified to suit other particular applications. The example chosen uses 
HIV Primer pair, SK145 and SK431 (Applied Biosystems), in conjuction with Applied 
Biosystem's GeneAmp 10X PCR Buffer II, MgCL 2 Solution, GeneAmp dNTPs, and 
PCR Carry-Over Prevention Kit, to amplify a 142-bp DNA fragment from the con- 
served gag region of HIV- 1 using the AmpliTaq Gold Hot Start process. 

1. 10X PCR Buffer II: 100 mm Tris-KCl, 500 mM KC1, pH 8.3 at room temperature. 

2. 25 mM MgCl 2 solution. 

3. dNTPs: 10 mM stocks of each of dATP, dCTP, dGTP; 20 mM stock of dUTP; all neutral- 
ized to pH 7.0 with NaOH. 

4. Primer 1: SK145. 25 mM in 10 mM Tris-HCl, pH 8.3 at room temperature. Sequence: 
5'-AGTGGGGGGACATCAAGCAGCCATGCAAAT-3'. 

5. Primer 2: SK431. 25 mM in 10 mM Tris-HCl, pH 8.3 at room temperature. Sequence: 
5-TGCTATGTCAGTTCCCCTTGGTTCTCT-3'. 

6. AmpErase® UNG: Uracil-N-glycosylase, 1.0 U/mL pH 8.3 at room temperature in 
150 mM NaCl, 30 mM Tris-HCl, pH 7.5 at room temperature, 10 mM ethylene- 
diaminetetraacetic acid (EDTA), 1.0 mM dithipthreitol (DTT), 0.05% Tween-20, 5% 
(v/v) glycerol. 

7. HIV-1 Positive Control DNA: 10 3 copies/mL in 10 mg/mL human placental DNA. 

8. AmpliTaq Gold: 5 U/mL. 

9. 0.5 mL microcentrifuge tubes (Applied Biosystems GeneAmp PCR microcentrifuge tubes). 
10. Thermal Cycler (PE Applied Biosystems GeneAmp PCR System). 

3. Methods 

3. 1. Hot Start Process 

In the AmpliTaq Gold Hot Start process (33), a master mix is prepared at room 
temperature, aliquoted into individual tubes, and thermal cycled. 



14 



Kolmodin and Birch 



1, Assemble the reagent mix as shown here: 



Reagent 


Volume (IX mix, uL) 


Final Concentration 
(per 100 uL Volume) 


Sterile Water 




N/A 


10X PCR Buffer II 


10.0 


IX 


25 void MgC12 


10.0 


2.5 mM 


10 mM dATP 


2.0 


200 \iM 


10 mM dCTP 


2.0 


200 uM 


10 mM dGTP 


2.0 


200 uM 


20 mM dUTP 


2.0 


400 uM 


25 uM Primer 1 (SK145) 


2.0 


50 pmol 


25iiMPrimer2(SK431) 


2.0 


50 pmol 


1 U/uL AmpErase UNG 


1.0 


1 .0 U/reaction 


10 3 copies/uL (+Control) 


0.1-10.0 


10 2 -10 4 copies/uL 


5 U/fiL AmpliTaq Gold 0.5 




2.5 U/reaction 


Total Volume 


100 u.L 





2. Add 100 uL of the above reagent mix to the bottom of each GeneAmp PCR reaction tube. 
Avoid splashing liquid onto the tube walls. If any liquid is present on the tube walls, spin 
the tube briefly in a microcentrifuge. 

3. Amplify the PCR amplifications within a programmed thermal cycler. For the Perkin 
Elmer DNA Thermal Cycler 9600, program and run the following linked files: 

a. CYCL File: 95°C for 9 min, 1 cycle; link to file (b). 

b. CYCL File: 94°C for 30 s, 60°C for 1 min, 43 cycles; link to file (c). 

c. CYCL File: 60°C for 10 min; 1 cycle; link to file (d). 

d. HOLD File: 10°C hold. 

3.2. Analysis of PCR Products (see Note 2) 

3.2.1. Agarose Gel Electrophoresis 

PCR products can be easily and quickly analyzed and resolved using a 3% NuSieve 
GTG agarose (FMC Bioproducts, Rockland, ME) and 1% Seakem GTG agarose (FMC 
Bioproducts) gel run in either TBE (89 mM Tris-borate, 2 mM EDTA) or TAE (40 mM 
Tris-acetate, 2 mM EDTA, pH approx 8.5). The resolved DNA bands are detected by 
staining the gels with either approx 0.5 u,g/mL of ethidium bromide, followed by 
destaining with water or SYBR® Green 1 (Molecular Probes Inc., Eugene, OR) and 
finally photographed under UV illumination. Use a 123-basepair (bp) or 1-kilobasepair 
(kbp) ladder as a convenient marker for size estimates of the products (34). 

3.2.2. Other Analytical Methods 

A variety of other detection methods are available for PCR product analysis, such 
as ethidium bromide-stained 8-10% polyacrylamide gels run in TBE buffer, Southern 
gels or dot/blots, subcloning and direct sequencing, HPLC analysis, and the use of 
96-well microplates, to name a few. The reverse dot-blot method combines PCR 
amplification with nonradioactive detection (35). 

The introduction of fluorescent dyes to PCR, together with a suitable instrument for 
real-time, online quantification of PCR products during amplification has led to the 
development of kinetic PCR or quantitative PCR. Quantitative PCR (QPC) measures 



PCR: Basic Principles 15 

PCR product accumulation during the exponential phase of the reaction and before 
amplification becomes vulnerable, i.e., when reagents become limited. The ABI Prism 
7700 (Applied Biosystems) and the LightCycler (Roche Molecular Biochemicals, 
Mannheim, Germany) are integrated fluorescent detection devices that allow fluores- 
cence monitoring either continuously or once per cycle. These instruments can also 
characterize PCR products by their melting characteristics, e.g., to discriminate single- 
base mutations from a wild-type sequence. The recently designed Mx4000™ Multi- 
plex Quantitative PCR System (Strategene, La Jolla, CA) can generate and analyze 
data for multiple fluorescent real-time QPCR assays. 

4. Notes 

1. Even though the PCR process has greatly enhanced scientific studies, a variety of prob- 
lems with the process, easily revealed by ethidium-bromides-stained agarose gel electro- 
phoresis, can and may need to be considered when encountered. For example, unexpected 
molecular weight size bands (nonspecific banding) or smears can be produced. These 
unexpected products accumulate from the enzymatic extension of primers that annealed 
to nonspecific target sites. Second, primer-dimer (approx 40-60 bp in length, the sum of 
the two primers) can be produced. Primer-dimer can arise during PCR amplification when 
the DNA template is left out of the reaction, too many amplification cycles are used, or 
the primers are designed with partial complementarity at their 3' ends. Note, an increase 
in primer-dimer formation will decrease the production of the desired product. Third, Taq 
DNA polymerase, which lacks the 3'-5' exonuclease "proofreading" activity, will occa- 
sionally incorporate the wrong base during PCR extension. The consequences of Taq 
misincorporations usually have little effect, but should be considered during PCR cloning 
and subsequent cycle sequencing. 

2. PCR amplification for user-selected templates and primers are considered "failures" 
when 1) no product bands are observed; 2) the PCR product band is multibanded; or 3) 
the PCR product is smeared. These "failures" can be investigated and turned into suc- 
cessful PCR by manipulation of a number of variables, such as enzyme and salt concen- 
trations, denaturation and anneal/extend times and temperatures, primer design, and 
hot start procedures (35). 

When no desired PCR product band is observed, initially verify the enzyme addition 
and/or concentration by titrating the enzyme concentration. Second, the magnesium ion 
concentration is also critical, so care should be taken not to lower the magnesium 
ion molarity on addition of reagents (i.e., buffers containing EDTA will chelate out the 
magnesium ion). The denaturation and anneal/extend times and temperatures may be too 
high or too low, causing failures, and can be varied to increase reaction specificity. 
Finally, the chemical integrity of the primers should be considered. In cases where the 
PCR product band is multibanded, consider raising the anneal temperature in increments 
of 2°C and/or review the primer design and composition. 

If a smear of the PCR product band is seen on an ethidium-bromide- stained agarose 
gel, consider the following options initially, individually, or in combination: decreasing 
the enzyme concentration, lowering the magnesium ion concentration, lengthening and/ 
or raising the denaturation time and temperature, shortening the extension time, reducing 
the overall cycle number, and decreasing the possibility of carryover contamination. 
Finally, in PCR amplifications where the PCR product band was initially observed, and 



16 Ko I mod in and Birch 

on later trials a partial or complete loss of the product bands is observed, consider testing 
new aliquots of reagents and decreasing the possibility of carryover contamination. 

For PCR amplifications using a modified DNA polymerase such as AmpliTaq Gold, 
poor product amplification can occur owing to inadequate activation of the Hot Start 
polymerase. Incubation time, temperature, and pH are critical for Hot Start polymerase 
activation. Contaminants added with the target, whether remnants from the sample's 
source or artifacts of the sample's preparation, can affect the PCR pH. Contaminants may 
also directly inhibit the polymerase. Hot Start polymerase activation begins during 
the pre-PCR activation step and continues through the PCR cycles' denaturation steps. 
The temperature and duration of these steps and the total number of PCR cycles should be 
optimized. Additional PCR cycles may increase specific product yield without increasing 
background in a Hot Start PCR. Raising the temperature above 95°C for any PCR step 
may irreversibly denature the polymerase. 

References 

1 . Innes, M. A., Gelfand, D. H., Sninsky, J. J. and White, T. J., eds. (1990) PCR Protocols, A 
Guide to Methods and Application, Academic, San Diego, CA. 

2. Mullis, K. B. and Faloonam F. A. (1987) Specific synthesis of DNA in vitro via a poly- 
merase chain reaction. Meth. Enzymol. 155, 335-350. 

3. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R„ Horn, G. T., et al. 
(1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA poly- 
merase. Science 239, 487-491. 

4. Saiki, R. K., Scharf, S. J., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and 
Arnheim, N. (1985) Enzymatic amplification of (3-globin genomic sequences and restric- 
tion site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354. 

5. Scharf, S. J., Horn, G. T., and Erlich, H. A. (1986) Direct cloning and sequence analysis of 
enzymatically amplified genomic sequences. Science 233, 1076-1087. 

6. Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Quantitation of mRNA by the poly- 
merase chain reaction. Proc. Na.t Acad. Sci. USA 86, 9717-9721. Nature 1, 

7. Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237, 238. 

8. Orrego, C. (1990) Organizing a laboratory for PCR work, in PCR Protocols. A Guide to 
Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., 
eds.), Academic, San Diego, CA, pp. 447-454. 

9. Kitchin, P. A., Szotyori, Z., Fromholc, C, and Almond, N. (1990) Avoiding false posi- 
tives. Nature 344, 201. 

10. Longo, N., Berninger, N.S., and Hartley, J. L. (1990) Use of uracil DNA glycosylase to 
control carry-over contamination in polymerase chain reactions. Gene 93, 125-128. 

11. Chou, Q., Russell, M., Birch D. E., Raymond, J., and Bloch, W. (1992) Prevention of pre- 
PCR mis-priming and primer dimerization improves low-copy-number amplifications. 
Nucl. Acids Res. 20, 1717-1723. 

12. Birch, D. E., Kolmodin, L., Laird, W. J„ McKinney, N., Wong, J., Young, K. K. Y., et al. 
(1996) Simplified Hot Start PCR. Nature 381, 445,446. 

13. Ailenberg, M. and Silverman, M. (2000) Controlled hot start and improved specificity in 
carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS). 
Biotechniques 29, 1018-1020, 1022-1024. 

14. Kaboev, O. K., Luchkina, L. A., Tret'iakov, A. N., and Bahrmand, A. R. (2000) PCR hot 
start using primers with the structure of molecular beacons (hairpin-like structure). Nucl. 
Acids Res. 28, E94. 



PCR: Basic Principles 1 7 

15. Kainz, P., Schmiedlechner, A., and Strack, H. B. (2000) Specificity-enhanced hot-start 
PCR: addition of double- stranded DNA fragments adapted to the annealing temperature. 
Biotechniques 28, 278-82. 

16. Dang, C. and Jayasena, S. (1996) Oligonucleotide inhibitors of Taq DNA polymerase 
facilitate detection of low copy number targets by PCR. J . Molec. Biol. 264, 268-278. 

17. Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. D. (1988) DNA sequenc- 
ing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain 
reaction-amplified DNA. Proc. Nat. Acad. Sci. USA 85, 9436-9440. 

18. Abramson, R. D. (1995) Thermostable DNA polymerases, in PCR Strategies (Innes, M. 
A., Gelfand, D. H., and Sninsky, J. J., eds.), Academic, San Diego, CA, pp. 39-57. 

19. Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991) Detection of 
specific polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of 
Thermus aquaticus DNA polymerase. Proc. Nat. Acad. Sci. USA 88, 7276-7280. 

20. Sobral, B. W. S. and Honeycutt, R. J. (1993) High output genetic mapping of polyploids 
using PCR generated markers. Theor. Appl. Genet. 86, 105-112. 

21. Myers, T. W. and Gelfand, D. H. (1991) Reverse transcription and DNA amplification by 
a Thermus thermophilus DNA polymerase. Biochemistry 30, 7661-7666. 

22. Myers, T. W. and Sigua, C. L. (1995) Amplification of RNA, in PCR Strategies (Innes, M. 
A., Gelfand, D. H., and Sninsky, J. J., eds.), Academic, San Diego, CA, pp. 58-58. 

23. Cheng, S., Fockler, C, Barnes, W. M., and Higuchi, R. (1994) Effective amplification of 
long targets from cloned inserts and human genomic DNA. Proc Nat Acad Sci USA 91, 
5695-5699. 

24. Cheng, S., Chen, Y., Monforte, J. A., Higuchi, R., and Van Houten, B. (1995) Template 
integrity is essential for PCR amplification of 20- to 30-kb sequences from genomic DNA. 
PCR Meth. Amplificat. 4, 294-298. 

25. Erlich, H. A., ed. ( 1989) PCR Technology, Principles and Application for DNA Amplifica- 
tion. Stockton, New York. 

26. Landre, P. A., Gelfand, D. H., and Watson, R. H. (1995) The use of cosolvents to enhance 
amplification by the polymerase chain reaction, in PCR Strategies (Innes, M. A., Gelfand, 
D. H., and Sninsky, J. J., eds.), Academic, San Diego, CA, pp. 3-16. 

27. Henke, W., Herdel, K., Jung, K. Schnorr, D., and Loening, S. (1997) Betaine improves the 
PCR amplification of GC-rich DNA sequences. Nucl. Acids Res. 25 (19), 3957-3958. 

28. Paabo, S., Gifford, J. A., and Wilson, A. C. (1988) Mitochondrial DNA sequences from a 
7000-year old brain. Nucl. Acids Res. 16, 9775-9787. 

29. Sarker G, Kapeiner, S., and Sommer, S. S. (1990) Formamide can drastically increase the 
specificity of PCR. Nucl. Acid Res. 18, 7465. 

30. Smith, K. T., Long, C. M., Bowman, B. and Manos, M. M. (1990) Using cosolvents to 
enhance PCR amplification. Amplifications 9/90 (5), 16,17. 

31. Kreader, C. (1996) Relief of amplification inhibition in PCR with bovine serum albumin 
or T4 gene 32 protein. Appl. Environ. Microbiol. 62, 1102-1106. 

32. Kovarova, M. and Draber, P. (2000) New specificity and yield enhancer of polymerase 
chain reactions. Nucl. Acids Res. 28, E70. 

33. AmpliTaq Gold. Package Insert. BIO-142, 54,670-3/96. Applied Biosystems, Foster 
City, CA. 

34. Sambrook, J., Fritsch, E. F., and Maniatis, T. eds (1989) Molecular Cloning: A Labora- 
tory Manual, 2nd ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, 
pp. 6.20, 6.21, B.23, B.24. 



18 Ko I mod in and Birch 

35. Saiki, R. K., Walsh, P. S., Levenson, C. H., and Erlich, H. A. (1989) Genetic analysis of 
amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc. Nat. 
Acad. Sri. USA 86, 6230-6234. 

36. Kolmodin, L., Cheng, S., and Akers, J. (1995) GeneAmp XL PCR Kit. Amplifications: A 
Forum for PCR Users (The Perkin-Elmer Corporation) 13, 1-5. 



Computer Programs for PCR Primer Design 
and Analysis 

Bing-Yuan Chen, Harry W. Janes, and Steve Chen 



1. Introduction 

1.1. Core Parameters in Primer Design 

1.1. 1. T m , Primer Length, and GC Content (GC %) 

Heat will separate or "melt" double-stranded DNA into single-stranded DNA by 
disrupting its hydrogen bonds. T m (melting temperature) is the temperature at which 
half the DNA strands are single-stranded and half are double-stranded. T m character- 
izes the stability of the DNA hybrid formed between an oligonucleotide and its comple- 
mentary strand and therefore is a core parameter in primer design. It is affected by 
primer length , primer sequence, salt concentration, primer concentration, and the pres- 
ence of denaturants (such as formamide or DMSO). 

All other conditions set, T m is characteristic of the primer composition. Primer with 
higher G+C content (GC %) has a higher T m because of more hydrogen bonds (three 
hydrogen bonds between G and C, but two between A and T). The T m of a primer also 
increases with its length. A simple formula for calculation of the T m (1 ,2) (see Note 1) is 

T m = 2 x AT + 4 x CG 

where AT is the sum of A and T nucleotides, and CG is the sum of C and G nucleotides 
in the primer. 

1.1.2. Primer Specificity 

Primer specificity is another important parameter in PCR primer design. To amplify 
only the intended fragment, the primers should bind to the target sequence only but not 
somewhere else. In other words, the target sequence should occur only once in the 
template. Primer length not only affects the T m , as discussed earlier, but also the 
uniqueness (specificity) of the sequence in the template (3). Suppose the DNA 
sequence is entirely random (which may not be true), the chance of finding an A, G, C, 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

19 



20 Chen, Janes, and Chen 

or T in any given DNA sequence is one quarter (1/4 1 ), so a 16 base primer will statis- 
tically occur only once in every 4 16 bases, or about 4 billion bases, which is about the 
size of the human genome. Therefore, the binding of a 16 base or longer primer with 
its target sequence is an extremely sequence-specific process. Of course, to be abso- 
lutely sure that the target sequence occurs only once, you would need to check the 
entire sequence of the template DNA, which is not possible in most cases. However, it 
is often useful to search the current DNA sequence databases to check if the chosen 
primer has gross homology with repetitive sequences or with other loci elsewhere in the 
genome. For genomic DNA amplification 17-mer or longer primers are routinely used. 

1.1.3. Primer Sequence and Hairpin (Self-Complementarity) 
and Self-Dimer (Dimer Formation) 

The hardest part in PCR primer design is to avoid primer complementarity, espe- 
cially at the 3' ends. When part of a primer is complementary to another part of itself, 
the primer may fold in half and form a so-called hairpin structure, which is stabilized 
by the complementary base pairing. The hairpin structure is a problem for PCR because 
the primer is interacting with itself and is not available for the desired reaction. Fur- 
thermore, the primer molecule could be extended by DNA polymerase so that its 
sequence is changed and it is no longer capable of binding to the target site. 

Similar to the hairpin structure, if not carefully designed, one primer molecule may 
hybridize to another primer molecule and acts as template for each other, resulting in 
primer-dimers. Primer-dimer formation causes the same problems to PCR reaction as 
the hairpin structure. It may also act as a competitor to amplification of the target 
DNA (4). Usually it is very hard and time-consuming to catch the hairpin structure or 
primer-dimer formation manually by a naked eye. However, they can be easily detected 
by primer analysis programs. 

1.2. General Rules for PCR Primer Design 

According to Innis and Gelfand (5) the rules for primer design is as follows: 

1. Primers should be 17-28 bases in length; 

2. Base composition should be 50-60% (G+C); 

3. Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and 
increases efficiency of priming; 

4. Tms between 55-80°C are preferred; 

5. Avoid primers with 3' complementarity (results in primer-dimers). 3'-ends of primers 
should not be complementary (i.e., basepair), as otherwise primer dimers will be 
synthesised preferentially to any other product; 

6. Primer self-complementarity (ability to form secondary structures such as hairpins) should 
be avoided; 

7. Runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or 
C-rich sequences (because of stability of annealing), and should be avoided. 

Because two different primers are needed for PCR reaction, primer-dimer forma- 
tion between the two primers should also be checked and avoided if possible. It is 
desirable that primer T m s should be similar (within 8°C or so). If they are too different, 
a suitable annealing temperature may be hard to find. At high annealing temperature, 



PCR Primer Design 21 

the primer with the lower T m may not work, whereas at low annealing temperature, 
amplification will be less efficient because the primer with the higher T m will 
misprime. 

In reality, primer selection is often empirical. It varies greatly from researcher to 
researcher in regard to the criteria they use. 

1.3. Computer Programs for PCR Primer Design and Analysis 

1.3. 1. Computer Programs for Nondegenerate PCR Primer Design 

For primer design, most researchers used to visually inspect target DNA sequence 
to find primer(s) with the characteristics they prefer, which are usually similar to the 
guidelines we mentioned earlier. As computers are widely used in molecular biology, 
a large number of computer programs have been specifically developed for 
nondegenerate primer selection, which makes the PCR primer design more efficient 
and reliable. Most sequencing analysis packages, such as Vector NTI (InforMax Inc.), 
usually contain a primer design module. In this chapter, we focus on free online (web) 
primer design programs (see Note 2). Selected computer programs for nondegenerate 
PCR primer design and their features are listed in Table 1. 

From a computational point of view the design of nondegenerate PCR primers is 
relatively simple: find short substrings from DNA nucleotide string that meet certain 
criteria. Although the criteria vary between programs, the core parameters, such as the 
primer length, T m , GC content, and self-complementarity, are shared by these programs. 

1.3.2. Computer Programs for Degenerate PCR Primer Design 

In the experiments to amplify the novel members of gene families or cognate 
sequences from different organisms by PCR, the exact sequence of the target gene is 
not known. We usually align all known sequences for this gene and find the most 
conserved regions, then design corresponding "degenerate" primers, which are a set of 
primers with nucleotide diversity at several positions in the sequence. Degeneracies 
obviously increase the chances of amplifying the target sequence but reduce the speci- 
ficity of the primer(s) at the same time. 

Designing degenerate primers has been considered more of an art than a science. 
There are much less computer programs for degenerate primer design (see Table 2) 
than for nondegenerate primer design. 

1.3.3. Computer Programs for Primer Analysis 

Even if you prefer to design primers by yourself, not by a computer program, it is 
advised that your primers should be analyzed by a computer program to determine T m , 
possible hairpin structure, primer-dimers, and other properties before you place the 
order for them. Table 3 lists two computer programs for this purpose. 

2. Materials 

1. Computer: A computer (PC or Macintosh) with high-speed internet access. 

2. Programs: Web Browser, Netscape (5.0 or above) or Internet Explorer (4.0 or higher). 

3. Input files for primer design: DNA sequence file DNA.txt (see Table 4) and protein 
sequence file Protein.txt (see Table 5) (see Note 4). 



Computer Programs for PCR Primer Design 
and Analysis 

Bing-Yuan Chen, Harry W. Janes, and Steve Chen 



1. Introduction 

1.1. Core Parameters in Primer Design 

1.1. 1. T m , Primer Length, and GC Content (GC %) 

Heat will separate or "melt" double-stranded DNA into single-stranded DNA by 
disrupting its hydrogen bonds. T m (melting temperature) is the temperature at which 
half the DNA strands are single-stranded and half are double-stranded. T m character- 
izes the stability of the DNA hybrid formed between an oligonucleotide and its comple- 
mentary strand and therefore is a core parameter in primer design. It is affected by 
primer length , primer sequence, salt concentration, primer concentration, and the pres- 
ence of denaturants (such as formamide or DMSO). 

All other conditions set, T m is characteristic of the primer composition. Primer with 
higher G+C content (GC %) has a higher T m because of more hydrogen bonds (three 
hydrogen bonds between G and C, but two between A and T). The T m of a primer also 
increases with its length. A simple formula for calculation of the T m (1 ,2) (see Note 1) is 

T m = 2 x AT + 4 x CG 

where AT is the sum of A and T nucleotides, and CG is the sum of C and G nucleotides 
in the primer. 

1.1.2. Primer Specificity 

Primer specificity is another important parameter in PCR primer design. To amplify 
only the intended fragment, the primers should bind to the target sequence only but not 
somewhere else. In other words, the target sequence should occur only once in the 
template. Primer length not only affects the T m , as discussed earlier, but also the 
uniqueness (specificity) of the sequence in the template (3). Suppose the DNA 
sequence is entirely random (which may not be true), the chance of finding an A, G, C, 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

19 



20 Chen, Janes, and Chen 

or T in any given DNA sequence is one quarter (1/4 1 ), so a 16 base primer will statis- 
tically occur only once in every 4 16 bases, or about 4 billion bases, which is about the 
size of the human genome. Therefore, the binding of a 16 base or longer primer with 
its target sequence is an extremely sequence-specific process. Of course, to be abso- 
lutely sure that the target sequence occurs only once, you would need to check the 
entire sequence of the template DNA, which is not possible in most cases. However, it 
is often useful to search the current DNA sequence databases to check if the chosen 
primer has gross homology with repetitive sequences or with other loci elsewhere in the 
genome. For genomic DNA amplification 17-mer or longer primers are routinely used. 

1.1.3. Primer Sequence and Hairpin (Self-Complementarity) 
and Self-Dimer (Dimer Formation) 

The hardest part in PCR primer design is to avoid primer complementarity, espe- 
cially at the 3' ends. When part of a primer is complementary to another part of itself, 
the primer may fold in half and form a so-called hairpin structure, which is stabilized 
by the complementary base pairing. The hairpin structure is a problem for PCR because 
the primer is interacting with itself and is not available for the desired reaction. Fur- 
thermore, the primer molecule could be extended by DNA polymerase so that its 
sequence is changed and it is no longer capable of binding to the target site. 

Similar to the hairpin structure, if not carefully designed, one primer molecule may 
hybridize to another primer molecule and acts as template for each other, resulting in 
primer-dimers. Primer-dimer formation causes the same problems to PCR reaction as 
the hairpin structure. It may also act as a competitor to amplification of the target 
DNA (4). Usually it is very hard and time-consuming to catch the hairpin structure or 
primer-dimer formation manually by a naked eye. However, they can be easily detected 
by primer analysis programs. 

1.2. General Rules for PCR Primer Design 

According to Innis and Gelfand (5) the rules for primer design is as follows: 

1. Primers should be 17-28 bases in length; 

2. Base composition should be 50-60% (G+C); 

3. Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and 
increases efficiency of priming; 

4. Tms between 55-80°C are preferred; 

5. Avoid primers with 3' complementarity (results in primer-dimers). 3'-ends of primers 
should not be complementary (i.e., basepair), as otherwise primer dimers will be 
synthesised preferentially to any other product; 

6. Primer self-complementarity (ability to form secondary structures such as hairpins) should 
be avoided; 

7. Runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or 
C-rich sequences (because of stability of annealing), and should be avoided. 

Because two different primers are needed for PCR reaction, primer-dimer forma- 
tion between the two primers should also be checked and avoided if possible. It is 
desirable that primer T m s should be similar (within 8°C or so). If they are too different, 
a suitable annealing temperature may be hard to find. At high annealing temperature, 



PCR Primer Design 21 

the primer with the lower T m may not work, whereas at low annealing temperature, 
amplification will be less efficient because the primer with the higher T m will 
misprime. 

In reality, primer selection is often empirical. It varies greatly from researcher to 
researcher in regard to the criteria they use. 

1.3. Computer Programs for PCR Primer Design and Analysis 

1.3. 1. Computer Programs for Nondegenerate PCR Primer Design 

For primer design, most researchers used to visually inspect target DNA sequence 
to find primer(s) with the characteristics they prefer, which are usually similar to the 
guidelines we mentioned earlier. As computers are widely used in molecular biology, 
a large number of computer programs have been specifically developed for 
nondegenerate primer selection, which makes the PCR primer design more efficient 
and reliable. Most sequencing analysis packages, such as Vector NTI (InforMax Inc.), 
usually contain a primer design module. In this chapter, we focus on free online (web) 
primer design programs (see Note 2). Selected computer programs for nondegenerate 
PCR primer design and their features are listed in Table 1. 

From a computational point of view the design of nondegenerate PCR primers is 
relatively simple: find short substrings from DNA nucleotide string that meet certain 
criteria. Although the criteria vary between programs, the core parameters, such as the 
primer length, T m , GC content, and self-complementarity, are shared by these programs. 

1.3.2. Computer Programs for Degenerate PCR Primer Design 

In the experiments to amplify the novel members of gene families or cognate 
sequences from different organisms by PCR, the exact sequence of the target gene is 
not known. We usually align all known sequences for this gene and find the most 
conserved regions, then design corresponding "degenerate" primers, which are a set of 
primers with nucleotide diversity at several positions in the sequence. Degeneracies 
obviously increase the chances of amplifying the target sequence but reduce the speci- 
ficity of the primer(s) at the same time. 

Designing degenerate primers has been considered more of an art than a science. 
There are much less computer programs for degenerate primer design (see Table 2) 
than for nondegenerate primer design. 

1.3.3. Computer Programs for Primer Analysis 

Even if you prefer to design primers by yourself, not by a computer program, it is 
advised that your primers should be analyzed by a computer program to determine T m , 
possible hairpin structure, primer-dimers, and other properties before you place the 
order for them. Table 3 lists two computer programs for this purpose. 

2. Materials 

1. Computer: A computer (PC or Macintosh) with high-speed internet access. 

2. Programs: Web Browser, Netscape (5.0 or above) or Internet Explorer (4.0 or higher). 

3. Input files for primer design: DNA sequence file DNA.txt (see Table 4) and protein 
sequence file Protein.txt (see Table 5) (see Note 4). 



22 



Chen, Janes, and Chen 



Table 1 

Selected Computer Programs for Nondegenerate PCR Primer Design 



Program 



Operating 
System 



Features 



URL (see Note 3) 



Oligos 



Oligo 



Windows 
9X/NT 



GCG Prime Unix 



Primer3 


Internet 




Browser 


Web Primer 


Internet 




Browser 


iOligo 


Windows 




9X/NT, 




Mac 


xprimer 


Internet 




Browser 


PCR Help! 


Windows 




9X/NT 



Windows 

9X/NT, 

Mac 



The Primer Internet 
Generator Browser 



Free download 

The program includes several tools: make complement, 

reverse complement and inverted strand; search the 

sequence; extract from selected sites. 

(Reference Lowe T 1990) 

Commercial 
Available within GCG 

This program selects primers according to a number 
of user-specified criteria including length, GC content, 
and annealing temperature. Potential primers can also 
be tested for self-complementarity and complementarity 
to each other to minimize the formation of primer 
dimers during the PCR. 

Free 

Lots of user-configurable parameters 

Primer design for both PCR and hybridization 

Nice interface with useful help pages 

Free 

Best for designing primers to clone yeast genes. 

Can use a standard yeast gene name or systematic yeast 

name as DNA source input 

Commercial 

Retrieval of Sequences from NCBI 

Sequence Editor 

Analysis of Oligonucleotide's Characteristics 

Submission of Oligo Orders by email 

Free 

The user can select repeat database and genome model. 

Nice graphical display of suggested primers 

Commercial 
Free Demo Download 

User-friendly "PCR Wizard" allows you to design 
primers to any given DNA template sequence as well 
as to generate a Techne Genius Thermal Cycler pro- 
gram file, which can be sent from a PC directly to 
multiple Genius thermal cyclers (up to 32) 

Commercial 

Free Demo Download 

Nice graphical interface for searching, selecting, and 

analyzing primers from known sequences 

Cross-compatible Multiplex PCR Primer Search 

Priming Efficiency Calculations 

Free 

Designs Site Directed Mutagenesis primers 

The program analyzes the original nucleotide sequence 

and desired amino acid sequence and designs a primer 

that either has a new restriction enzyme site or is 

missing an old one. This allows for faster sorting out 

of mutated and nonmutated sequences. 



http://www.biocenter.- 
helsinki.fi/bi/bare- 1_ 
html/oligos.htm 



http://www.gcg.com/ 
products/wis-pkg- 
programs.html 
#Primer 



http://www-genome. 
wi.mit.edu/cgi-bin/ 
primer/primer3_ 
www.cgi 

http://genome- 
www2.stanford.edu/ 
cgi-bin/SGD/web- 
primer 

http://www.caesar 
software.com/pages/ 
products/ioligo/ 
ioligo.shtml 

http://alces.med.umn. 
edu/webprimers.html 

http://www.techneuk. 

co.uk/CatMol/ 

pcrhelp.htm 



http://www.oligo.net/ 



http://www.med.jhu. 
edu/medcenter/ 
primer/primer. cgi 



PCR Primer Design 



23 



Table 2 

Selected Computer Programs for Degenerate PCR Primer Design 



Program 



Operating 
System 



Features 



URL 



GeneFisher 


Internet 


Free 




Browser 


Processes aligned or unaligned 
sequences of DNA or protein 


CODEHOP 


Internet 


Free 




Browser 


Design degenerate PCR primers from 
protein multiple sequence alignments. 
The multiple-sequence alignments 
should be of amino acid sequences of 
the proteins and be in the Blocks 
Database format 


Primer 


Mac and 


Commercial 


Premier 5 


Windows 


Free Demo Download 




9X/NT 


Reverse translate a protein sequence 



and design primers in regions of 
low degeneracy 



http://bibiserv.techfak. 
uni-bielefeld.de/genefisher/ 

http://www. blocks. 
fhcrc.org/codehop.html 



http://www.premierbiosoft 
http://www.premierbiosoft 
com/primerdesign/ 
primerdesign.html 



Table 3 

Selected Computer Programs for PCR Primer Analysis 



Program 



Operating 
System 



Features 



URL 



Oligo 


Internet 


Free 


Analyzer 


Browser 


Caculate Tm, find possible primer 
hairpin structure and primer dimer 
formation, Blast search databases 
for primer homologs 


NetPrimer 


Internet 


Free 




Browser 


Java Applet 



Analyze basic properties and 
second structures for an individual 
primer or primer pair. Also give a 
primer rating and a report of the 
analysis results 



http://playground.idtdna.com/ 
program/oligocalc/oligocalc.asp 



http://www.premierbiosoft. 
com/netprimer/netprimer.html 



3. Methods 

3.1. Designing Nondegenerate PCR Primers Using Primer3 

Primer3 was developed at Whitehead Institute for Biomedical Research and Howard 
Hughes Medical Institute. It contains so many parameters that most people only need 
a subset of them to use as the criteria for primer selection. 

3.1.1. Design Primers with the Default Settings 

Primer3 provides default values for core parameters (see Table 6 for a selected list. 
Go to Primer3 web page for a complete list and their meanings). If these default set- 
tings meet your needs, then use the following method to select your primers. 



24 Chen, Janes, and Chen 

Table 4 

Input File DNA.txt 

1 GGGGAAGTGC AATCACACTC TACCACACAC TCTCTATAGT ATCTATAGTT GAGAGCAAGC 
61 TTTGTTAACA ATGGCGGCTT CCATTGGAGC CTTAAAATCT TCACCTTCTT CCCACAATTG 
121 CATCAATGAG AGAAGAAATG ATTCTACACG TGCAATATCC AGCAGAAATC TCTCATTTTC 
181 GTCTTCTCAT CTCGCCGGAG ACAAGTTGAT GCCTGTATCG TCCTTACGTT CCCAAGGAGT 
241 ACGATTCAAT GTGAGAAGAA GTCCATTGAT TGTGTCTCCT AAGGCTGTTT CTGATTCGCA 
301 GAATTCACAG ACATGTCTGG ATCCAGATGC TAGCAGGAGT GTTTTGGGAA TTATTCTTGG 
361 AGGTGGAGCT GGGACCCGAC TTTATCCTCT AACTAAAAAA AGAGCAAAAC CTGCGGTTCC 
421 ACTTGGAGCA AATTATCGTC TGATTGACAT TCCCGTAAGC AATTGCTTGA ACAGTAACAT 
481 ATCCAAGATC TATGTTCTCA CACAATTCAA CTCTGCCTCT CTAAATCGCC ACCTTTCACG 
541 GGCATATGCT AGCAATATGG GAGAATACAA AAACGAGGGC TTTGTGGAAG TTCTTGCTGC 
601 TCAACAAAGT CCGGAGAACC CCGATTGGTT CCAGGGCACT GCGGACGCTG TCAGACAATA 
661 TCTGTGGTTG TTTGAGGAGC ATAATGTTCT TGAATACCTT ATACTTGCTG GAGATCATCT 
721 GTATCGAATG GATTATGAAA AGTTTATTCA AGCCCACAGG GAAACAGATG CTGATATTAC 
781 TGTTGCCGCA CTGCCAATGG ACGAGAAGCG TGCCACTGCA TTCGGTCTCA TGAAGATTGA 
841 CGAAGAAGGA CGCATTATTG AATTTGCAGA GAAACCGCAA GGAGAGCAAC TGCAAGCAAT 
901 GAAAGTGGAT ACTACCATTT TAGGTCTTGA TGACAAGAGA GCTAAAGAAA TGCCTTTTAT 
961 CGCCAGTATG GGTATATATG TCATTAGCAA AGACGTGATG TTAAACCTAC TTCGTGACAA 
1021 GTTCCCTGGG GCCAATGATT TTGGTAGTGA AGTTATTCCT GGTGCAACTT CACTTGGGAT 
1081 GAGAGTGCAA GCTTATTTAT ATGATGGGTA CTGGGAAGAT ATTGGTACCA TTGAAGCTTT 
1141 CTACAATGCC AATTTGGGCA TTACAAAAAA GCCGGTGCCA GATTTTAGCT TTTACGACCG 
1201 ATCAGCCCCA ATCTACACCC AACCTCGATA TTTGCCACCT TCAAAAATGC TTGATGCCGA 
1261 TGTCACAGAT AGTGTCATTG GTGAAGGTTG TGTGATCAAG AACTGTAAGA TTCACCATTC 
1321 CGTGGTTGGG CTCAGATCAT GCATATCAGA GGGAGCAATT ATAGAAGACT CACTTTTGAT 
1381 GGGGGCAGAT TACTACGAGA CTGATGCTGA GAGGAAGCTG CTGGCTGCAA AGGGCAGTGT 
1441 CCCAATTGGC ATCGGCAAGA ATTGTCTATA CAAAAGAGCC ATTATCGACA AGAATGCTCG 
1501 TATAGGGGAC AATGTGAAGA TCATTAACAA AGACAATGTT CAAGAAGCGG CTAGGGAAAC 
1561 AGATGGATAC TTCATCAAGA GTGGGATCGT CACTGTCATC AAGGATGCTT TGATTCCAAG 
1621 TGGAATCGTC ATTTAAAGGA ACGCATTATA ACTTGGTTGC CCTCCAAGAT TTTGGCTAAA 
1681 CAGCCATGAG GTACAAACGT GCCGAAGTTT TATTTTCCTA TGCTGTAGAA ATCTAGTGTA 
1741 CATCTTGCTT TTATGATACT TCTCATTACC TGGTTGCTGT AAAAATTATT CGTCTAAAAT 
1801 AAAAATAAAT CTACCATTAC ACCA 



1. Start a web browser (Netscape or Internet Explorer). 

2. Replace the default URL address with http://www-genome.wi.mit.edu/cgi-bin/primer/ 
primer3_www.cgi and hit return. After connection Primer3 web page will appear in your 
browser. 

3. Open the DNA sequence input file DNA.txt (see Note 5) using your favorite text editor, 
such as Notepad in Windows, then copy the sequence by going to Edit/Select All, Edit/ 
Copy in the menubar. Close file DNA.txt. 

4. In your browser click on the top sequence input box, then paste the above sequence by 
going to Edit/Paste in the menubar. 

5. Click Pick Primers Button (there are six Pick Primers buttons on the page. Click any one 
of them will do the same). After a few s/min, Primer3 Output will be returned. The top 
part of the output is shown in Table 7. 

The other parts of the output not shown are: whole input sequence and arrows, 
which nicely indicate the location of the primers above; additional four primer pairs; 
and statistics about the primer selection process. 



PCR Primer Design 25 

Table 5 

Input File Protein.txt 

>Protein 1 

MKSTVHLGRVSTGGFNNGEKEIFGEKIRGSLNNNLRINQLSKSL 

KLEKKIKPGVAYSVITTENDTETVFVDMPRLERRRANPKDVAAVILGGGEGTKLFPLT 

SRTATPAVPVGGCYRLIDIPMSNCINSAINKIFVLTQYNSAALNRHIARTYFGNGVSF 

GDGFVEVLAATQTPGEAGKKWFQGTADAVRKFIWVFEDAKNKNIENILVLSGDHLYRM 

DYMELVQNHIDRNADITLSCAPAEDSRASDFGLVKIDSRGRVVQFAENQRFELKAMLV 

DTSLVGLSPQDAKKSPYIASMGVYVFKTDVLLKLLKWSYPTSNDFGSEIIPAAIDDYN 

VQAYIFKDYWEDIGTIKSFYNASLALTQEFPEFQFYDPKTPFYTSPRFLPPTKIDNCK 

IKDAIISHGCFLRDCTVEHSIVGERSRLDCGVELKDTFMMGADYYQTESEIASLLAEG 

KVPIGIGENTKIRKCIIDKNAKIGKNVSIINKDGVQEADRPEEGFYIRSGIIIISEKA 

TIRDGTVI 

>Protein2 

MDALCAGTAQSVAICNQESTFWGQKISGRRLINKGFGVRWCKSF 

TTQQRGKNVTSAVLTRDINKEMLPFENSMFEEQPTAEPKAVASVILGGGVGTRLFPLT 

SRRAKPAVPIGGCYRVIDVPMSNCINSGIRKIFILTQFNSFSLNRHLARTYNFGNGVG 

FGDGFVEVLAATQTPGDAGKMWFQGTADAVRQFIWVFENQKNKNVEHIIILSGDHLYR 

MNYMDFVQKHIDANADITVSCVPMDDGRASDFGLMKIDETGRIIQFVEKPKGPALKAM 

QVDTSILGLSEQEASNFPYIASMGVYVFKTDVLLNLLKSAYPSCNDFGSEIIPSAVKD 

HNVQAYLFNDYWEDIGTVKSFFDANLALTKQPPKFDFNDPKTPFYTSARFLPPTKVDK 

SRIVDAIISHGCFLRECNIQHSIVGVRSRLDYGVEFKDTMMMGADYYQTESEIASLLA 

EGKVPIGVGPNTKIQKCIIDKNAKIGKDVVILNKQGVEEADRSAEGFYIRSGITVIMK 

NATIKDGTVI 



Table 6 

Selected Default Settings for Primer3 

Parameter Minimum Optimum Maximum 

Primer size (base pairs) 

Primer T m (°C) 

Max T m Difference (°C) 

Primer GC% 

Product size (basepairs) 



Table 7 
Primer3 Output 

WARNING: Numbers in input sequence were deleted. 

No misprinting library specified 

Using 1 -based sequence positions 

OLIGO start len tm gc% any 3' seq 

LEFT PRIMER 890 20 59.99 45.00 4.00 0.00 ctgcaagcaatgaaagtgga 

RIGHT PRIMER 1090 20 59.83 50.00 4.00 2.00 ttgcactctcatcccaagtg 

SEQUENCE SIZE: 1824 

INCLUDED REGION SIZE: 1824 

PRODUCT SIZE: 201, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 3.00 



18 


20 


27 


57 


60 


63 

100 


20 




80 


00 


200 


1000 



26 Chen, Janes, and Chen 

3.1.2. Design Primers with User-Defined Settings 

Often the default values need to be altered because they do not meet a researcher's 
needs or Primer3 did not find an appropriate PCR primer pair. The following are help- 
ful guidelines for adjusting these parameters if Primer3 failed to select a primer: 

a. Adjust location: pick a wider range to examine and allow for longer product size; 

b. Change primer size: usually easier to find compatible primers if they are shorter; 

c. Lower primer T m . 

Because there are so many configurable parameters in Primer3, it is impossible to 
explain their uses and try to change them here. Fortunately, the default values need not 
to be altered for most parameters. The readers should read the Primer3 help page and 
understand the uses of the parameters before trying to change them. 

In the following method, we will try to design primers to clone the coding region in 
DNA.txt, which is from nucleotide 71 to 1636. 

1. Start a web browser (Netscape or Internet Explorer). 

2. Replace the default URL address with http://www-genome.wi.mit.edu/cgibin/primer/ 
primer3_www.cgi and hit return. After connection, Primer3 web page will appear in your 
browser. 

3. Open the DNA sequence input file DNA.txt using your favorite text editor, such as 
Notepad in Windows, then copy the sequence by going to Edit/Select All, Edit/Copy in 
the menubar. Close file DNA.txt. 

4. In your browser, click on the top sequence input box, then paste the above sequence by 
going to Edit/Paste in the menubar. 

5. Type 71,1565 in the Targets input box. Change Product Size/Max from 1000 to 1824, 
then click Pick Primers button. (There are six Pick Primers buttons on the page. Click 
any one of them will do the same). After a few s/min, Primer3 Output will be returned. 
Are there any primers returned? 

3.2. Designing Degenerate PCR Primers Using GeneFisher 

GeneFisher is an interactive degenerate primer design software. The current ver- 
sion, GeneFisher 1.22, processes aligned or unaligned sequences of DNA or protein. 
In the following method, we will use two unaligned protein sequences as the sequence 
input and design degenerate primers which could amplify the cDNAs encoding these 
two proteins and their related family members (if any). 

1. Start web browser (.see Note 6). 

2. Replace the default URL address with http://bibiserv.techfak.unibielefeld.de/genefisher/ 
and hit return. After connection, GeneFisher Interactive PCR Primer Design home 
page will appear in your browser. 

3. Click Start button on the page. After a few s/min, the Interactive Primer Design inter- 
face will be open. 

4. In the User Data area of the page, type your E-mail ID and Project name. Click this 
button in the Sequence Data area to clear the sample sequence, then copy the two protein 
sequences from Protein.txt and paste to the Sequence Data area (see Note 7). Click OK 
button in the Submit Query area to accept your choice. 



PCR Primer Design 27 

5. After a few s/min GeneFisher Sequence Input page will appear. Check that the protein 
lengths match with the input sequences. Click OK button to accept the two protein 
sequences. 

6. After GeneFisher Alignment Status page returns, click OK button to use ClustalW as 
the alignment tool. ClustalW Multiple Sequence Alignment Setup page will appear. Click 
OK button to accept the default parameters. 

7. Click Progress button on the GeneFisher Clustal Alignment page, which will open a 
new browser window. Click Reload button repeatedly in the new window to check the 
status of the alignment. If the last line on the page shows "GDE-Alignment file created" 
(The alignment time depends on your input. It takes several minutes for Protein.txt.) Then 
click Alignment button in the original window, which will show the alignment results in 
a new window. 

8. We are satisfied with the alignment results, so go to the original window and click the 
Consensus button. Sequence Consensus page will return. Click OK button to accept the 
default consensus parameters, which will open the GeneFisher Consensus page. 

9. Click Progress button to check the consensus calculation progress. If you are satisfied 
with the consensus calculation, click Consensus button on the GeneFisher Consensus 
page, which will show the alignment results in a new window. Click Go! button in the 
original window to generate primers. 

10. After a few s/min, Primer Design page will appear. Click OK button to accept the default 
settings for primer design, which will open GeneFisher Primer Calculation page. Wait 
a few s/min, then click Results button, which will open the Primer Calculation Results 
page. Unfortunately, the results show that no primer pairs were generated. The rejection 
statistics underneath give some clues on why the primer selection fails. Click the Redo 
button to return to the Primer Design page. 

11. Make the following changes to the primer parameters: 

a. Set primer length from 15 to 22 bp. 

b. Set GC content from 35 to 85%. 

c. Set melting temperature T m from 42 to 65°C. 

d. Set product size from 100 to 1500 bp. 

e. Set primer degeneracy 512-fold. 

f. Set 3' GC content from 35 to 85%. 

Repeat the primer design step above (step 10). This time, seven primer pairs were 
returned (see Table 8). If you click the primer sequence link (Forward Primer or Reverse 
Primer), the GeneFisher Primers Profile - Data Sheet about that primer pair will 
be returned in a new window. If you click the primer position link (FPPos. RPPos.), the 
Textual Primer Pair Visualization of that primer pair will be shown in a new window. 

3.3. Analyze PCR Primers Using NetPrimer 

1. Start a web browser (Netscape or Internet Explorer). 

2. Replace the default URL address with http://www.premierbiosoft.com/netprimer/ 
netprimer.html and hit return. After connection, NetPrimer web page will appear in your 
browser. 

3. Click the click here link in the page to launch the NetPrimer applet. 

4. After the applet is launched, type the following sequence in the Oligo Sequence input 
area: ctgcaagcaatgaaagtgga, then click the Analyze button. The analysis results of the 
primer, such as T m , molecular weight, GC%, rating, and stability, will be shown in the 



28 



Chen, Janes, and Chen 



Table 8 

GeneFisher Output (IUB Code for Sequence) 



7 best Pairs (of max. 7) 



ID 



Forward Primer 



Reverse Primer 



Prod. 
Qual. Len. T m Diff. FPPos. RPPos. 



1 NTAYMGNATGRAYTAYATGGA 

2 NTAYMGNATGRAYTAYATGGA 

3 NTAYMGNATGRAYTAYATGGA 

4 NTTYAANGAYTAYTGGGA 

5 NTTYAANGAYTAYTGGGA 

6 NTAYMGNATGRAYTAYATGGA 

7 NTAYMGNATGRAYTAYATGGA 



GTyTGrTArTArTCnGCnCCCA 

TyTGrTArTArTCnGCnCCCAT 

AynGTnCCdATrTCyTCCCA 

GTyTGrTArTArTCnGCnCCCA 

TyTGrTArTArTCnGCnCCCAT 

GTyTTrAAnACrTAnACnCCCA 264 

TyTTrAAnACrTAnACnCCCAT 



659 


653 


6 


653 


1306 


658 


652 


5 


653 


1305 


398 


388 


6 


653 


1041 


290 


278 


12 


1028 


1306 


289 


277 


11 


1028 


1305 


264 


248 


6 


653 


901 


262 


247 


5 


653 


900 



Results area of the applet. You may also click the following buttons: Hairpin, Dimer, 
Palindrome, and Repeat & Run, to check the corresponding properties about the primer. 

4. Notes 

1 . This formula only gives a very approximate T m in the absence of denaturating agents such 
as formamide and DMSO, and it is only valid for primers < 20 nucleotides in length. For 
PCR purposes T m -5°C is a good annealing temperature to start with. However, optimal 
annealing temperatures can only be determined experimentally for a certain primer/tem- 
plate combination and there is no formula currently available to accurately define their 
relationships. 

For longer primers, the nearest-neighbor method (6) offers a reliable estimation of the 
T m and its formula is the following: 

T m = AH / (A + AS + R x ln[C/4]) - 273.15 + 16.6 x log[salt] 

where: 

AH (cal/mole) is the sum of the nearest-neighbor enthalpy changes for DNA helix 
formation (<0). 

A (cal/degree Celsius/mole) is a constant for helix initiation, which is equal to 
-10.8 cal /degree Celsius/mole for nonself-complementary sequences and = -12.4 for 
self-complementary sequences. 

AS (cal/degree Celsius/mole)is the sum of the nearest-neighbor entropy changes 
for helix formation (<0). 

R is the molar gas constant (1.987 cal/degree Celsius/mole). 

C is the primer concentration. 

[salt] is the salt concentration. 

However, primer design programs may use different formula to calculate T m . For 
example, the Primer3 program uses the following formula: 

T m = 81.5 + 16.6(loglO([Na+])) + 0.41 x (%GC) - 600/length 

where [Na+] is the molar sodium concentration, (%GC) is the percent of Gs and Cs in 
the sequence, and length is the length of the sequence. 



PCR Primer Design 29 

2. Keep in mind that internet is not a secure place to send your sequences. If you care about 
the privacy of your sequences or do not have internet access, then download a freeware or 
buy a commercial package and use them instead to design your primers. 

3. URL stands for Universal Resource Locator, which is a unique address on the internet. 
However, URL is quite dynamic. Old web sites could be shut down and new sites could 
be set up, resulting in change of web address for a particular page or disappearance of a 
web page. Try to search for the new address of a web page by using a search engine, such 
as www.google.com. 

4. You can also use your own in-house sequences for primer designs discussed in the Meth- 
ods section. Of course, the results will vary. 

5. For the input sequence of Primer3, numbers and blanks are ignored. Other letters are 
treated as N. FASTA format is acceptable. It is assumed that the strand direction is 5'->3'. 

6. Although GeneFisher system has been optimized for Netscape Navigator version 4.x and 
above, our testing showed that it works fine with Internet Explorer 5.0 and above when 
protein sequences are used as inputs. Netscape Navigator should be used when DNA 
sequences are used as inputs. 

7. If you use your own input sequences, make sure that the sequences have significant 
homology. Otherwise the primer pair which meets your parameters will be very hard to 
find, if not impossible. Do not use the Browse... button to load your sequences, as it 
appears that there is a bug in reading the sequences from a file by GeneFisher. 

References 

1. Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K. (1979) 
Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of 
single base pair mismatch. Nucl. Acids Res. 6, 3543-3557. 

2. Rychlik, W. and Rhoads, R. E. (1989) A computer program for choosing optimal oligo- 
nucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucl. 
Acids Res. 17,8543-8551. 

3. Wu, D. Y., Ugozzoli, L., Pal, B. K., Qian, J., and Wallace, R. B. (1991) The effect of 
temperature and oligonucleotide primer length on the specificity and efficiency of ampli- 
fication by the polymerase chain reaction. DNA Cell Biol. 10, 233-238. 

4. Rychlik, W. (1993) Selection of primers for polymerase chain reaction, in PCR Protocols. 
Current Methods and Applications (White, B. A., ed.), Humana, Totowa, NJ, pp. 31-40. 

5. Innis, M. A. and Gelfand, D. H. (1990) Optimization of PCRs, in PCR Protocols (Innis, M. 
A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic, New York, pp. 3-12. 

6. Breslauer, K. J., Frank, R., Blocker, H., and Markey, L. A. (1986) Predicting DNA duplex 
stability from the base sequence. Proc. Natl. Acad. Sci. USA 83, 3746-3750. 



Single-Step PCR Optimization 

Using Touchdown and Stepdown PCR Programming 



Kenneth H. Roux 



1. Introduction 

Polymerase chain reaction (PCR) optimization and troubleshooting can consume 
considerable energy and resources because of the finicky and often unpredictable 
nature of the reactions. Small variations in any of the many variables in a given reac- 
tion can have a pronounced effect on the resultant amplicon profile. Reactions that are 
too stringent yield negligible product, and reactions that are not stringent enough yield 
artifactual amplicons. Variables include concentrations of Mg 2+ , H + , dNTPs, primers, 
and template, as well as cycling parameters. Regarding the latter, the value selected 
for the annealing temperature is most critical. Unfortunately, even with the most 
sophisticated algorithms (i.e., OLIGO) it is often difficult to predict the amplification 
optima, a priori leaving no other choice but to employ empirical determination. 

Touchdown (TD) PCR (1,2) and its sister technique, stepdown (SD) PCR (2) repre- 
sent a markedly different approach that, in a single amplification regimen, inherently 
compensates for suboptimal reagent concentrations and less than perfect cycling 
parameters. Rather than guessing (or using imprecise calculations) to arrive at an appro- 
priate temperature for the primer extension segment of the cycle, one can cast a wider 
net by using progressively lower annealing temperatures over consecutive cycles. The 
goal is to select a broad range of annealing temperatures that begins above the esti- 
mated melting temperature (T m ) and ends below it (see Note 1). Typically, one runs a 
TD PCR program at 2 cycles/°C declining over a 10-20°C range at 1°C intervals. In 
this way, the first primer-template hybridizations and primer extensions will be those 
with the highest specificity, i.e., presumably, the combination that gives the desired 
amplicon. Although the annealing temperature continues to drop in subsequent cycles to 
levels that normally would promote spurious amplification, the desired product, hav- 
ing already experienced several cycles of amplification, will be in a position to out- 
compete most lower T m (spurious) amplicons. If, for example, there is only a 3°C 



From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 



31 



32 Roux 

difference between the T m of the target amplicon and the T m of the first-primed spurious 
amplicon, the desired product will have undergone up to a 64-fold (2 6 ) amplification. 

Our experience has been that TD PCR is applicable to a wide range of PCR situa- 
tions (2,3). At one extreme, TD PCR generally yields a single strong amplicon from 
genomic DNA even when the primer-template combinations are grossly mismatched 
(see Note 2). Mismatching might occur when attempting to amplify specific members 
from a complex multigene family, when using nucleotide sequence information 
deduced from an amino acid sequence, or when amplifying across species lines (4-6). 
Primer-template basepair mismatches are permissible and can even be near (but prob- 
ably not at) the 3' end of the primer (3). TD PCR can also compensate for suboptimal 
buffer composition (e.g., Mg 2+ concentration) (2). On the other hand, reactions that 
are already optimal, as assessed by conventional PCR, will usually yield equally strong 
amplicons even when using a broad temperature range TD PCR protocol in which the 
annealing temperature dips well below the T m . Stated another way, TD PCR appears to 
greatly aid marginal reactions while not imposing significant penalties on already 
robust reactions. Hence, TD PCR can be used routinely in lieu of conventional PCR 
and need not be viewed solely as an optimization procedure (7). 

One potential drawback to TD PCR stems from the complexity of the programming 
on some thermal cyclers (see Note 3). Because of the numerous (10 to 20) annealing 
temperatures used, a large segment of the programming capacity of conventional ther- 
mal cyclers can be encumbered. Also, attempts to adjust the annealing temperature 
range can involve considerable reprogramming (see Note 4). Some newer thermal 
cyclers circumvent these problems by permitting the programming of automatic incre- 
mental temperature changes in progressive cycles. We have tested modified versions 
of TD PCR, which we term SD PCR, that utilize simplified programming (2). For SD 
PCR, one uses fewer, but larger, annealing temperature steps with proportionately 
more cycles per step. For example, a program might consist of three or four steps, at 
three to four cycles per step, with 3-5°C temperature differences between steps. SD 
PCR is not quiet as universally applicable as TD PCR, but is adequate for many appli- 
cations. The ease in programming may frequently be worth the trade-off. 

2. Materials 

2. 1. Touchdown PCR with Mismatched Primer-Template Pairs 

1. Template DNA (rabbit genomic liver 125 ng/uL). 

2. Primers. Stock solutions are at 200 (xg/mL in H 2 0. The following primer pairs yield a 
445-bp amplicon. The sites of mismatches are in capital letters. The sequence of the cor- 
responding genomic homologous strand (shown in brackets) are for comparative pur- 
poses (see Note 5 for comments on degenerate primer design). 

a. Primers: 5'cttgccaGtaatatAcgccctgcTaaCTtg3'; 5'ggatcttctgttgatgtctgactGttGgAg 3' 

b. Homologous genomic sequences: [5'cttgccaAtaatatCcgccctgcCaaTCtg 3']; 5'ggatct- 
tctgttgatgtctgactAttTgTg 3' 

3. 10X PCR buffer: 500 mM KC1, 100 mM Tris-HCl, pH 9.0, 1% Triton X-100, 2 mM of 
each dNTP (A, T, C, and G), and 15 mM MgCl 2 (see Note 6). 

4. AmpliTaq DNA Polymerase (Perkin Elmer, Norwalk, CT). 

5. Sterile mineral oil. 



Single-Step PCR Optimization 33 

6. Standard wall 0.6-mL capped conical tubes. 

7. Equipment and reagents for 1.5% agarose gel electrophoresis. 

2.2. Stepdown PCR with a Mismatched Degenerate Primer 

1. Template DNA (rabbit genomic liver 125 ng/uL). 

2. Primers: Prepare stock solutions at 200 ug/mL. The following primer pair yields a 703-bp 
amplicon. The sites of mismatches are in capital letters. Degeneracies are separated by a 
slash and are in parenthesis. The sequence of the genomic homologous strand correspond- 
ing to the primer with degeneracies is presented (shown in brackets) for comparative 
purposes. 

a. Primers: 5'agggatcgggtgaaaggggtctcagc3'; 5'ttAtgagcattcat(a/G)aacttctggagg3'. 

b. Homologous genomic sequence: [5'agggatcgggtgaaaggggtctcagc3']; [5'ttGtgagcattcat- 
Aaacttctggagg 3']. 

3. 10X PCR buffer: 500 mM KC1, 100 mM Tris-HCl, pH 9.0, 1% Triton X-100, 2 mM of 
each dNTP (A, T, C, and G), and 15 mM MgCl 2 . 

4. AmpliTaq DNA Polymerase. 

5. Equipment and reagents for 1.5% agarose gel electrophoresis. 

3. Methods 

Programming of the thermal cycler for TD and SD PCR are described first, fol- 
lowed by specific PCR conditions used for both reactions. 

3.1. TD PCR Programming 

1. Set thermal cycler to denature for 1 min at 94°C, anneal for 2 min, and primer extend for 
3 min at 74°C. 

2. Follow the cycling program with a 7-min primer extension step and a 4°C soak step (see 
Note 7). 

3. Set the annealing stage for 2 cycles/ c C beginning at 55°C and decreasing at 1°C increments 
to 41°C (i.e., 30 total cycles in 15 steps) to be followed by 10 additional cycles at 40°C. 

3.2. SD PCR Programming 

1. Set thermal cycler to denature for 1 min at 94°C, anneal for 2 min, and primer extend for 
3 min at 74°C as detailed later. Follow the cycling program by a 7-min primer extension 
step and a 4 C C soak step (see Note 7). Program the annealing stage for six cycles per 
temperature step beginning at 70°C and decreasing at 3°C increments to 58°C (i.e., 30 
total cycles in five steps) to be followed by 10 additional cycles at 55°C (see Note 8 for 
programming considerations). 

2. Analyze and reamplify as described later. 

3.3. PCR Setup 

1. Set up master mix for 50-uL reactions as indicated in Table 1. 

2. Dispense master mix to 0.6-mL standard wall PCR tubes. Add 50 (xL mineral oil to each 
tube and place in thermal cycler (Perkin Elmer DNA Thermal Cycler). Begin initial cycle 
and add 2.5 uE (1.25 U) of a 1:10 dilution of polymerase to each tube only after the 
temperature exceeds 80°C in the thermal cycler (i.e., the hot start protocol, see Note 9). 
Cap tubes and continue cycling. 

3. Following amplification, monitor results by running 3 uE on a 1.5% agarose/ethidium 
bromide gel and view by UV illumination. If a product is not evident or the desired 



34 



Roux 



Table 1 

PCR Master Mix 





Stock 


Amount 




Final 


Components 


concentration 


per 50 u,L reaction, 


|.iL 


concentration 


dNTP mix 


2.0 mM ea. 




5 




0.2 mM 


PCR buffer 


10X 




5 




IX 


Primer 1 


200 ng/uL 




1 




4.0 ng/[xL 


Primer 2 


200 ng/uL 




1 




4.0 ng/uL 


Template 


125 ng/|xL 




3 




7.5 ng/(xL 


MgCl 2 


25 mM 




3 




1.5 mM 


H 2 


— 


Subtotal: 


29.5 

47.5 








amplicon is of insufficient amount, amplify for an additional 5-10 cycles (with TD PCR, 
you are not sure exactly how many nonproductive and suboptimal cycles preceded the start 
of efficient amplification) at 5°C below the lowest annealing temperature previously used. 
4. If the desired product is still not evident, consider conventional or TD nested PCR on a 
1 : 100 to 1 : 1000 dilution of the initial TD PCR reaction. 

4. Notes 

1. The T m for the primer-template combination can be roughly estimated using the formula: 



r m =2(A + T) + 4(G + C) 



(1) 



For primer-template combinations with known or suspected mismatches, 5-20°C should 
be subtracted from the normal annealing temperature. Of course, more sophisticated pro- 
grams, such as OLIGO Primer Analysis Software (National Biosciences, Inc., Plymouth, 
MN) (10,11), may also be used to calculate the T m . 

An estimation of the T m is particularly difficult when using primers and templates con- 
taining mismatched base pairs. We have successfully amplified, to a single intensely stain- 
ing band, primer-template pairs containing three to five mismatches with the template 
(3). In all cases, we used a TD PCR program in which the annealing temperatures dropped 
from 55-41°C at 2 cycles/°C. 

If using a thermal cycler that has a programmable automatic temperature variation fea- 
ture, set the annealing stages to decline by 0.5°C/cycle. For a standard thermal cycler, 
program 2 cycles/°C drop (for example, cycles 1 and 2, 65°C; cycles 3 and 4, 64°C, and 
so forth). In both instances, the TD portion of the program should be followed by 10 
cycles at a fixed annealing temperature about 10 C C below the estimated T m . One should 
bear in mind that for situations in which the template is not fully complementary to the 
primers, once amplification commences, the amplicon will be fully complementary to 
the primers and thus will have a greater T m than initially estimated. On the other hand, 
we have noted that final stage amplification at 10°C or more below the estimated T m 
(rather than the 4-5°C usually recommended for standard PCR) can significantly 
increase the yield of otherwise marginal reactions (2). Avoid the temptation of adding 
too many cycles to this terminal fixed annealing temperature stage of the program. 
Excessive cycling can degrade the product and lead to spurious banding and high 
molecular weight smearing (14). 



Single-Step PCR Optimization 35 

4. A convenient way to adjust the TD temperature range segment of a conventional thermal 
cycler (i.e., having linked or sequential file programming) is to program files covering a 
wide range of annealing temperature (20-25 c C). The specific subset of files to be used in 
any given amplification protocol can be incorporated by simply linking the initial 5 min 
denaturation file to the file having the highest annealing temperature to be used and link- 
ing the file containing the lowest annealing temperature in the TD range to the terminal 
primer extension file. Be sure to keep note of these changes because they must be undone 
before the next alteration in the range. If this approach will tie up many files, you may 
wish to reprogram those files containing the segments to be deleted from the range (say 
5°C from the bottom) and using the freed file capacity to add new files to the top (begin- 
ning) of the program. Again, the initial denaturation stage and terminal primer extension 
steps must be linked to the beginning and end of the new range, respectively. Thermal 
cyclers in which individual files cannot be linked but which rely on a single long multi- 
step program are even less versatile and may require complete reprogramming. Fortu- 
nately, TD and SD PCR are very forgiving and given temperature range protocol can be 
applied to a wide variety of situations. 

5. Design the best primer set based on the information available. When designing primers to 
amplify genes of uncertain complementarity, try to cluster the sequence of greatest cer- 
tainty near the 3' ends of the primers. Degeneracy derived from multiple nucleotides or 
inosine residues at positions of uncertainty are permissible (4,5,8) but not necessary. Note 
that some polymerases other than Taq cannot prime from inosine-containing primers (9). 

6. Because of the minimal effort involved, it is generally advantageous to vary one of the 
buffer components (usually Mg 2+ ) during the initial optimization. 

7. Most time and temperature characteristics of the cycling program (denaturation, primer 
extension) will be the same as conventional PCR for your system if using primer-tem- 
plate combinations other than the examples described. 

8. The temperature range of SD PCR may be divided into three to five more or less equal 
increments (steps) and be programmed accordingly. If you can afford to tie up the pro- 
gramming capacity of the thermal cycler, more steps are better than fewer. Proportion the 
total number of cycles to be used in the SD segment of the program equally among the 
steps. Add 10 cycles at a fixed temperature well below the T m as described earlier. When 
background problems are expected to be minimal, a simple two step SD PCR protocol 
can still be advantageous. Here, the initial stage has perhaps six to 10 cycles at a fixed 
temperature 5°C above that which would normally be used in standard PCR (i.e., slightly 
above the calculated T m ). Even though a full 30 cycles at this elevated temperature would 
not be expected to yield a detectable amplicon, we have found (2) that sufficient, highly 
specific, amplification is occurring to allow the desired amplicon to dominate the ampli- 
fication throughout the remainder of the standard temperature cycles, thus reducing the 
possibility that unwanted amplicons will be generated. 

9. Because TD PCR is based on the use of high temperature to prevent spurious priming, 
it is imperative that hot start procedures be followed (12,13). If multiple samples are to 
be run, add an extended denaturation step to the beginning of the program or use the 
hold option. 

References 

1. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S. (1991) 'Touch- 
down' PCR to circumvent spurious priming during gene amplification. Nucl. Acids Res. 
19, 4008. 



36 Roux 

2. Hecker , K. H. and Roux, K. H. (1996) High and low annealing temperatures increase both 
specificity and yield in TD and SD PCR. BioTechniques 20, 478-485. 

3. Roux, K. H. (1994) Using mismatched primer-template pairs in TD PCR. BioTechniques 
16,812-814. 

4. Knoth, K., Roberds, S. Poteet, C, and Tamkun, M. (1988) Highly degenerate inosine- 
containing primers specifically amplify rare cDNA using the polymerase chain reaction. 
Nucl. Acids Res. 16, 10,932. 

5. Patil, R. V. and Dekker, E. E. (1990) PCR amplification of an Escherichia coli gene using 
mixed primers containing deoxyinosine at ambiguous positions in degenerate amino acid 
codons. Nucl. Acids Res. 18, 3080. 

6. Batzer, M. A., Carlton J. E., and Deininger, P. L. (1991) Enhanced evolutionary PCR 
using oligonucleotides with inosine at the 3'-terminus. Nucleic Acids Res. 19, 5081. 

7. Roux, K. H. (1995) Opitimization and troubleshooting in PCR. PCR Meth. Applicat. 4, 
S185-S194. 

8. Peterson, M. G., Inostroza, J., Maxon, M. E., Flores, O., Adomon, A., Reinberg, D., and 
Tjian, R. (1991) Structure and functional properties of human general transcription factor 
HE. Nature 354, 369-373. 

9. Knittel, T. and Picard, D. (1993) PCR with degenerate primers containing deoxyinosine 
fails with Pfu DNA polymerase. PCR Meth. Applicat. 2, 346-347. 

10. Rychlik, W. and Spencer, W. J. (1989) A computer program for choosing optimal oligo- 
nucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucl. 
Acids Res. 17,8543-8551. 

1 1 . Rychlik, W. (1994) New algorithm for determining primer efficiency in PCR and sequenc- 
ing. J. NIHRes. 6, 78. 

12. D' Aquila, R. T., Bechtel, L. J., Viteler, J. A., Eron, J. J., Gorczyca, P., and Kaplin, J. C. 
(1991) Maximizing sensitivity and specificity of PCR by preamplification heating. Nucl. 
Acids Res. 19, 3749. 

13. Erlich, H. A., Gelfand, D., and Sninsky, J. J. (1991) Recent advances in the polymerase 
chain reaction. Science 252, 1643-1651. 

14. Bell, D. A. and DeMarini, D. (1991) Excessive cycling converts PCR products to random- 
length higher molecular weight fragments. Nucl. Acids Res. 19, 5079. 



XL PCR Amplification of Long Targets 
from Genomic DNA 

Lori A. Kolmodin 
1. Introduction 

Long polymerase chain reaction (PCR) >(l-5), specifically, XL PCR (Extra-Long 
Polymerase Chain Reaction), has enabled amplification of expanded trinucleotide 
repeats of the neuromuscular disease myotonic dystrophy (6) a 9-kb HIV-1 pro virus 
from primary isolate DNA (7), 24.2-kb fragments from nanogram quantities of 
genomic DNA for DNA damage repair (8), and up to 42 kb of human genomic DNA 
(4). The capability of the long PCR process stems through the use of: 

1. Two thermophylic polymerases, one of which is highly processive (5' to 3' polymerase 
activity) and the other one possesses proofreading activity (3'-5' exonuclease activity) 
known to improve fidelity by removing mismatch bases that can cause pauses in poly- 
merization or termination of strand synthesis (2); 

2. Reduced denaturation time at moderately high temperatures aimed at protecting the DNA 
template against damage; 

3. An alkaline tricine buffer suggested to protect DNA from being nicked at elevated tem- 
peratures as a result of acidic conditions; 

4. Cosolvents such as dimethyl sulfoxide (DMSO) and glycerol known to stabilize enzymes 
(9), lower secondary structure, and lower melting and strand separation temperatures (1); 

5. Increased extension time to allow the polymerase to complete strand synthesis and to 
lower the frequency of recombination between partial extension products; 

6. Consideration of the integrity of the template (1). 

Primer design and reaction stringency must also be considered for successful single- 
copy gene amplifications from complex genomic DNA. Optimal reaction conditions 
are system dependent, particularly as the target length increases. With increasing 
understanding of the key PCR parameters and subsequent improvements in PCR pro- 
cess, increasingly longer targets will be routinely amplified. 

This ability to amplify longer targets by PCR has numerous applications. For exam- 
ple, larger steps may be taken in gene mapping and sequencing efforts. Amplification 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

37 



38 Kolmodin 

of increasingly larger segments of any gene can also enable the simultaneous char- 
acterization of several regions of interest, for both research and diagnostic purposes. 
XL PCR protocols may facilitate studies of apparently unclonable regions, and of 
certain viral genomes or recombinant phage X clones that are not readily cultured 
(3,5). XL PCR can also complement cloning approaches by providing larger quanti- 
ties of longer inserts. 

2. Materials 

2. 1. Preparation of Genomic DNA (see Notes 1 and 2) 

1 . Source of Template DNA, such as cultured cells available from the NIGMS Human Genetic 
Mutant Cell Repository (see Coriell Institute for Medical Research, Camden, NJ). 

2. Phosphate-buffered saline or other balanced salt solution. 

3. 10 mM Tris-HCl, pH 8.2, 0.4 M NaCl, 2 mM ethylenediamine tetracetic acid (EDTA). 

4. 10% sodium dodecyl sulfate (SDS). 

5. Proteinase K (2 mg/mL). Prepare fresh in 1% SDS, 2 mM EDTA. 

6. RNase A (10 mg/mL). Heat inactivated at 95(-100°C for 20 min. 

7. Saturated NaCl (approx 6 M). 

8. 95% (v/v) ethanol. 

9. 70% (v/v) ethanol. 

10. TE buffer: 10 mM Tris-HCl, pH 7.5-8.0, 1 mM EDTA. 

2.2. Analysis of Template Integrity 

1. Agarose (see Subheading 2.3.). 

2. 50 mM NaCl, 1 mM EDTA. 

3. 10X Alkaline running buffer: 0.5 N NaOH, 10 mM EDTA. 

4. 6X Ficoll/bromocresol green solution: 0.3 N NaOH, 6 mM EDTA, 18% Ficoll (type 400 
from Pharmacia, Piscataway, NJ, or Sigma, St. Louis, MO), 0.15% bromocresol green, 
0.25% xylene cyanol FF. 

5. 0.1 M Tris-HCl, pH 8.0, 1 mM EDTA. 

6. Ethidium bromide solution: 0.5 ug/mL in TAE buffer (40 mM Tris-acetate, 2 mM EDTA, 
pH approx 8.5). 

2.3. Primers 

Use primers designed with high specificity (see Subheading 3.3. and Note 3). 
Primer sequences for the 7.5 kb and 17.7 kb of the human (3-globin gene cluster and 
16.3 kb of the mitochondrial genome are listed in Note 4. Nontemplate sequences can 
be added at the ends of primers to utilize differential display methodology for detect- 
ing changes in gene expression and for subsequent restriction digestion and cloning 
(see Notes 4 and 5). Primers with one to two phosphorothioate bonds at the 3'-teminal 
internucleotide linkage can be employed to minimize primer degradation (see Notes 6 
and 7) (10,11). 

2.4. PCR Amplification 

1. GeneAmp XL PCR Kit (Applied Biosystems, Foster City, CA) consisting of: rTth DNA 
polymerase, XL; XL PCR Buffer II; dNTP blend; Mg(OA) 2 ; and control template and 
primers for amplification of a 20.8-kb sequence from the phage X genome (see Notes 8 
and 9). 



XL PCR Amplification of Long Targets 39 

2. AmpliWax PCR Gem 100s (Applied Biosystems). 

3. Perkin Elmer GeneAmp PCR Instrument System and PCR tubes with which the XL PCR 
reagents have been optimized and quality control tested (e.g., Perkin Elmer GeneAmp 
PCR System 9600 with 0.2 mL MicroAmp reaction tubes [Applied Biosystems]). 

2.5. Product Analysis 

1. SeaKem GTG agarose or SeaKem Gold agarose (FMC Bioproducts, Rockland, ME). 

2. TAE buffer: 40 mM Tris-acetate, 2 mM EDTA, pH 8.5 or TBE buffer: 89 mM Tris borate, 
2 mM EDTA. 

3. Ethidium bromide (10 mg/mL). Store in the dark. Wear gloves when handling. 

4. Restriction endonucleases. 

5. Micron- 100 spin column (Amicon, Berverly, MA) or Qiaquick PCR Purification Kit 
(QIAGEN, Chatsworth, CA). 

3. Methods 

3. 1. Preparation of Total Genomic DNA 

In general, the template DNA must be of good quality. The DNA should have mini- 
mal single- stranded nicks and double-stranded breaks within the long PCR template 
sample, particularly for targets longer than 15 kb. Successful amplification of long 
targets depends on the presence of intact copies of the single-stranded template, and 
the intactness of a DNA sample will depend on the method of preparation used. The 
method of choice may vary with the target and the source of template DNA (see Notes 
1 and 2). The procedure of Miller et al. (12), based on a high-salt extraction of pro- 
teins, has been adapted for isolation of DNA from cultured cells (1) as described in the 
following steps: 

1. Use a hematocytometer to determine the cell density. Freshly grown cells should be kept 
on ice until used. Cells may also be frozen in growth medium with 5% (v/v) DMSO for 
future use. 

2. Rinse the cells once with phosphate-buffered saline (PBS) or other balanced salt solution. 

3. Thoroughly resuspend a pellet of 1-3 x 10 6 cells with 0.6 mL of 10 mM Tris-HCl, pH 8.2, 
0.4 M NaCl, 2 mM EDTA in a 1.5-mL microcentrifuge tube. 

4. Add 40 uL of 10% SDS, 100 uL of 2 mg/mL proteinase K and 7.5 uL of RNase A. 

5. Mix thoroughly by inverting the tube several times or by vortexing briefly. 

6. Incubate each sample for at least 1 h, but preferably overnight, in a 50°C shaking water 
bath to digest the proteins. Complete digestion is critical for efficient recovery of DNA 
in Step 7. 

7. Add 0.2 mL saturated NaCl, shake the tube vigorously for 15 s, then microcentrifuge at 
10,000 rpm (7000g) for 15 min. The precipitated proteins should form a tight, white pel- 
let. If the pellet is not tight, repeat the microcentrifuge step. Incomplete separation of the 
proteins from the DNA will result in incomplete proteinase K/SDS treatment. 

8. Carefully divide the clear supernatant containing the DNA into two new microcentrifuge 
tubes, then add a 2x volume of 95% (v/v) Ethanol at room temperature to each sample 
and gently invert each tube several times to mix well. 

9. Centrifuge the tubes at 10,000g for 10-15 min to pellet the precipitated DNA. 

10. Remove the supernatant and rinse the pellet with 0.5-1.0 mL of 70% (v/v) ethanol. Drain 
the excess fluid onto absorbent paper and allow the pellet to air dry for 5-10 min before 



40 Kolmodin 

resuspension (see step 11). If the DNA becomes too dry, it will be difficult to resuspend 
in solution. If the amount of recovered DNA is sufficiently high, the precipitated DNA 
aggregate can be lifted out of the tube (using the tip of a Pasteur pipet that has been 
reshaped to form a hook with a Bunsen burner flame), rinsed in 70% ethanol, and air- 
dried for 5 min. 

11. Based on expected yields (e.g., approx 15-20 (xg total human genomic DNA from approx 
3 x 10 6 cells), resuspend the genomic DNA in TE buffer to a final concentration of 
<300 ng/uL. Avoid highly concentrated DNA solutions that can be heterogeneous and 
too viscous to handle easily. The precipitated DNA may require overnight incubation at 
4 C C to fully solubilize. Do not vortex the sample to aid in resuspending the DNA, because 
the shearing forces may introduce double-stranded breaks. 

12. Determine the DNA concentration from the absorbance reading (between 0.2 and 0.8, for 
accuracy) at 260 nm. If accurate DNA concentrations are critical, and residual RNA is 
present, a method based on ethidium bromide fluorescence is described in Note 10. 

13. Store genomic DNA stocks in TE buffer at 4°C to minimize the introduction of nicks 
through repeated freeze-thaw cycles. Alternatively, divide each stock across several tubes 
and freeze. Thaw aliquots as needed and store working stocks at 4°C. 

3.2. Analysis of Template Integrity (Optional) 

The single- stranded integrity of a template DNA preparation can be qualita- 
tively assessed using alkaline agarose gel electrophoresis, essentially as in ref. 13. 
Use 0.3-0.5% agarose gels to visualize from 2 to > 30-kb single-stranded DNA (see 
Note 11). 

1 . Prepare a molten agarose solution in 50 m/W NaCl, 1 raM EDTA. 

2. When the solution has cooled to approx 50°C, add O.lX-volume of 10X alkaline running 
buffer. Swirl to mix and then pour the gel. 

3. Presoak the solidified gel in IX alkaline running buffer for 30 min to ensure pH equilibration. 

4. Load samples with a 6X Ficoll/Bromocresol green solution (see Subheading 2.2.4. or 
ref. 13). 

5. Run the gel at 0.5-1.8 V/cm (e.g., 3.5-5 h) using a peristaltic pump to circulate the buffer. 
The buffer may become quite warm. Both the buffer level and gel position should be 
checked periodically during the run. 

6. Neutralize the gel by gently shaking in 0.1 M Tris-HCl, pH 8.0, 1 mM EDTA for 30 min, 
and then stain with approx 0.5 [ig/mL ethidium bromide in TAE buffer. 

3.3. Primer Design 

As with standard PCR, successful primers for the XL PCR process need to be deter- 
mined empirically. Certain guidelines are to be considered when designing the primer 
sequences for optimal reaction specificity. 

1. Choose primers with higher, balanced melting temperatures (T m ) of approx 62-70°C to 
allow the use of relatively high annealing temperatures (65-70°C). Sequences of 20-24 
bases can work well if the G+C-content is sufficiently high (50-60%), but longer primers 
(25-30 bases) may be needed if the A+T content is higher. 

2. Primer sequences should not be complementary within themselves or to each other. 
Regions of complementarity, particularity at the 3' end, may result in "primer-dimer" or 
"smeared" products (see Note 5). 



XL PCR Amplification of Long Targets 4 1 

3. Primers with balanced T m s within 1-2°C of each other are more likely to have the same 
optimal annealing temperature. If the difference in T m is >3°C, the primer with the higher 
T m may anneal to secondary primer sites during incubation at the lower temperature opti- 
mal for the second primer and contribute to nontarget products. 

4. Primers used in standard PCR, generally designed with annealing temperatures of 5°C 
below the T m (14), may also work at higher annealing temperatures, particularly when 
longer incubation times are used. 

5. Primers that can be used for control XL PCR amplifications with human genomic DNA 
are listed in Note 4. 

6. Software programs to calculate melting temperatures (include Oligo 5.0 (National Bio- 
sciences, Plymouth, MN) and Melt (in BASIC, from J. Wetmur, Mt. Sinai School of 
Medicine, NY), Primer Premier 5 (Premier Biosoft International), and PrimerSelect (from 
DNAStar)(jee Note 12). Wu et al. (15) have developed an algorothm for an Oligo- 
nucleotide's "effective priming temperature" (T p ) based on its "effective length" (Ln): 

T p C = 22 + lA6Ln (1) 

Ln = 2 (number of G and C bases) + (number of A and T bases) (5). 

7. In any PCR, nontarget sites within the genome that have sufficient complementarity to 
the 3' end of a primer sequence can be secondary priming sites. As target length increases, 
however, secondary priming sites within the target are also likely. Shorter secondary prod- 
ucts are likely to be more efficiently amplified than a long target, and thus may accumu- 
late at the expense of the desired product. Consequently, specificity at the primer 
annealing step is critical for successful amplification of long targets. 

Whenever possible, candidate primers for XL PCR should be screened against avail- 
able sequence databases, particularly against any known sequences within the target 
and related loci (e.g., for gene families). Avoid primers within interspersed repetitive 
elements, such as Alu sequences (16). The program Oligo (see Subheading 3.3.6.) 
can be used to scan a template sequence for potential secondary priming sites. Right 
Primer 1.01 (BioDisk Software, San Francisco, CA) can be used to screen sequences 
deposited in GeneBank (National Institutes of Health) for various target genomes to 
estimate the relative frequency of selected primer sequences. Web sites such as NCBI 
offers BLAST for doing sequence homology searches online (see Note 12). 

3.4. PCR Amplification 

The GeneAmp XL PCR Kit (see Note 8) is designed to use the Ampliwax PCR 
Gem-facilitated hot start process (17). In this process, a solid wax layer is formed over 
a subset of PCR reagents (e.g., the lower reagent mix containing 30-50% of the total 
reaction volume), with the remaining reagents (e.g., the upper reagent mix containing 
the remaining 50-70% of the total reaction volume) added above the wax layer. Dur- 
ing the temperature ramp to the first denaturation step, the wax layer melts and is 
displaced by the upper reagent mix, which is more dense. Thermal convection suffices 
to completely mix the combined lower and upper reagent mixes, and the melted wax 
layer serves as a vapor barrier throughout the PCR amplification. Manual hot start 
processes can also be used (see Note 13), although reproducibility may be lower. The 
wax-mediated process also helps to minimize contamination between samples. 



42 Kolmodin 

The protocol below describes XL PCR amplification of a human genomic DNA 
target (e.g., using the primers RH1024 and RH1053 as in Note 4) and a volume ratio 
of the lowenupper reagent mixes equal to 40:60. Each mix can be assembled as a 
master mix sufficient for multiple reactions which allows for volume losses during 
aliquoting (e.g., a 10X master mix for nine reactions). 

1 . Assemble the lower reaction mix as listed in Table 1. For a 100-uL reaction, place 40 (xL 
of this lower reagent mix into the bottom of each MicroAmp reaction tube. Avoid splash- 
ing liquid onto the tube wall. If any liquid is present on the tube wall, spin the tube briefly 
in a microcentrifuge. 

2. Carefully add a single AmpliWax PCR Gem 100 to each tube containing the lower reagent 
mix (see Note 17). Melt the wax by incubating the reaction tubes at 75-80°C for 3-5 min. 
Allow the tube to cool to room temperature (or on ice) in order to solidify the wax layer. 

3. Assemble the upper reaction mix as listed in Table 2. For a 100-uL reaction, aliquot 60 uL 
of this upper reagent mix to each room temperature tube, above the solidified wax layer. 
Avoid splashing liquid onto the tube walls. If any liquid is present on the tube wall, tap 
the tube lightly to collect any droplets into the upper reagent layer. Do not spin the tubes 
in a microcentrifuge, as this will dislodge the wax layer. 

4. Amplify the PCR amplifications in a programmable thermal cycler (see Notes 24). For 
the Perkin Elmer GeneAmp System 9600, program the following method: 

a. HOLD: 94°C for 1 min (reagent mixing and initial template denaturation); 

b. CYCL: 94°C for 15 s (denaturation, see Note 25) and 68°C for 12 min (annealing 
and extension, see Notes 26-28 for 20 cycles; 

c. AUTO: 94°C for 15 s and 68°C for 12 min, adding 15 s per cycle (see Note 28) for 
17 cycles (see Note 29); 

d. HOLD: 70-72°C for 10 min (final completion of strand synthesis); 

e. HOLD: 4°C until tubes are removed (use the "Forever" software option). 

3.5. Product Analysis 

1 . To withdraw a sample, gently insert a pipet tip through the center of the solid wax layer to 
form a small hole. If the tip becomes plugged during this procedure, use a fresh tip to 
withdraw the reaction sample. 

2. The presence of PCR products can be quickly determined using a 0.6% SeaKem GTG 
agarose gel, run in either TAE or TBE buffer and stained in approx 0.5 (xg/mL ethidium 
bromide solution (see Notes 30-32). 

3. High-molecular weight products can be more accurately sized with a 0.3% SeaKem Gold 
agarose gel (see Note 11) run in TAE buffer and stained in approx 0.5 (xg/mL ethidium 
bromide solution. Depending on the level of resolution needed, run the gel at 7 V/cm for 
2 min and then either at 0.8 V/cm for up to 15 h or at 1 .5 V/cm for up to 6 h or at 5 V/cm 
for 1-2 h. Products may also be analyzed by pulse field gel electrophoresis (see Note 32). 

4. In general, XL PCR products may be analyzed directly by restriction digestion. If further 
manipulations, such as ligation and cloning, are planned, the reactions should be treated 
to remove the unincorporated dNTPs, primers and the tTth DNA polymerase, XL. If the 
polymerase and dNTPs are present during restriction digestions, recessed 3' termini may 
be filled in as they are created, eliminating such sites for ligation. Approaches to remove 
buffer components and unused primers is to use a Microcon Spin- 100 column or a Qiagen 
Qiaquick PCR Purification Kit (see Note 33). 



XL PCR Amplification of Long Targets 



43 



Table 1 

Guidelines for Preparation of the Lower Reagent Mix 





Volume, 


Final Concentation 


Reagent 


IX Mix, ixL 


per 100 U.L Volume 


Sterile Water 


14.0-15.2 


N/A 


3.3X XL Buffer II 


12.0 


IX 


10 mMdNTP Blend 


8.0 


800 \lM (200 \iM each dNTP) 
(see Note 14) 


25 mM MgCl 2 


4.4-5.2 


1.1-1.3 mM(see Note 15) 


25 \iM Primer RH 1024 


0.4-0.8 


0.1-0.2 uM, 

10-20 pmol/reaction 

(see Note 16) 


25 \iM Primer RH 105 3 


0.4-0.8 


0.1-0.2 uM, 

10-20 pmol/reaction 

(see Note 16) 


Total Volume 


40.0 uL 





Table 2 

Guidelines for Preparation of the Upper Reagent Mix 





Volume, 


Final Concentation 


Reagent 


IX Mix, uL 


per 100 uL Volume 


Sterile Water 


1.0-40.0 


N/A 


3.3X XL Buffer II 


18.0 


IX 


2 U/uL xTth DNA Polymerase, XL 


1.0 


2 U/reaction (see Note 18) 


Human Genomic DNA 


1.0^*0.0 


Up to (see Notes 19-23) 


Total Volume 


60.0 uL 





4. Notes 

1 . A number of other methods are available to prepare highly intact DNA. As discussed in 
ref. 1, these include the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN), 
Q1AGEN Genomic-tips (Chatsworth, CA), phenol-extraction (as in ref. 13, with high 
quality phenol), and Megapore dialysis (18). The Puregene DNA Isolation Kit is based on 
high-salt extraction of the proteins. It is important to fully resuspend the cell pellet with 
the lysis solution for the highest yields of DNA. The method does call for vigorous 
vortexing to mix the high-salt solution with the cell lysate. The benefits of this 15-20 s 
vortexing step for efficient precipitation of proteins and subsequent recovery of DNA 
outweigh the potential for damaging the DNA. In general, however, template stocks for 
long PCR should be handled gently. The QIAGEN Genomic-tips, based on an anion- 
exchange resin, are particularly useful for isolation of cosmid DNA without the use of 
organic solvents. To avoid clogging the Genomic-tip, take care to not load too many cells 



44 Kolmodin 

and to thoroughly resuspend the dilute sample with buffer QBT, as recommended, before 
loading the sample onto the column. The Megapore method (18) utilizes dialysis through 
type HA 0.45 [xm membranes (Millipore, Bedford, MA) to remove denatured proteins 
and cellular debris. This approach is designed to generate very high-molecular weight 
DNA fragments for cloning. 

2. A similar method that was not tested by the authors is the salt/chloroform extraction 
method by Mullenback et al. (19). The addition of chloroform facilitates the separation 
between the DNA and protein phases. 

3. Purification of primers by polyacrylamide gel electrophoresis does not appear to be gen- 
erally necessary, although significant levels of truncated sequences could contribute to 
priming at nonspecific, secondary sites. 

4. Three positive control primer sets for XL PCR with human genomic DNA are listed in 
Table 3 (20,21). These primers have been designed for use with a 67-68°C annealing tem- 
perature; at lower temperatures (e.g., 62°C), secondary products will also accumulate. The 
16.3-kb human mitochondrila genome primers can be used to confirm the presence of low- 
copy DNA because the mitochondrial genome is present in many copies per cell (22). 
Longer products are likely to be lower in yield because of lower reaction efficiencies. Cer- 
tain sequences may also be difficult to amplify regardless of the target length, perhaps 
because of their base composition and potential to form secondary structures. Consequently, 
the best controls for a troublesome system may be a series of primer pairs (e.g., one con- 
stant, the others variable in position) that define increasingly larger subsets of the target. 

5. In utilizing the Differential Display methodology, primers with 3' end modifications and 
5' end arbitrary primers can be introduced into long PCR amplifications. This technique 
should enable isolation of cDNAs that contain both the 3' untranslated transcript region 
and parts of the 3' end coding region, thus generating highly reproducible primer-set spe- 
cific fingerprints (23). 

6. For subsequent cloning of PCR product, primers can be used to introduce recognition 
sites for restriction endonucleases. Such sites would be added to the 5'-end of the target- 
binding sequence, with an additional GC-rich "5'-clamp" of several bases for efficient 
binding of the restriction endonucleases and subsequent digestion. These nontemplate 
bases at the 5'-end of the primer should not noticeably affect reaction specificity, but 
should be accounted for in determining the annealing temperature for the first few cycles 
(before the entire sequence has been incorporated into the template population). 

7. Primers synthesized with a single or double phosphorothioate (sulfur) linkage between 3' 
most bases has been shown to improve amplification by preventing digestion by the exo- 
nuclease activity of Vent DNA polymerase (10). 

8. If designing primers with mismatches to the template within the primer sequence, and 
intend to use a proofreading DNA polymerase, one must consider the position of the 
mismatch carefully. Due to the tendency of the proofreading polymerse to degrade single- 
stranded DNA (primer) one base at a time from the 3' end during PCR amplification, 
some of the primer may be degraded past the positions containing the desired base changes 
before the primer anneals to the template. However, primers designed and synthesized 
with phosphorothioate linkages on the last two 3' bases will render the primer extendable 
but not degradable (11). 

9. XL PCR buffer II is composed of tricine to maintain a protective pH during thermal 
cycling and the cosolvents glycerol and DMSO to effectively lower melting and strand 
separation temperatures. The 3'-5' exonuclease, "proofreading," activity of rTth DNA 
polymerase, XL facilitates the completion of strand synthesis in each cycle by removing 



XL PCR Amplification of Long Targets 45 



Table 3 

Positive Control Primer Sets 



Complements 




61986-62007 




48528-48550 


13.5 kb 


44348-44369 


17.7 kb 


Compliments 




14816-14841 




15149-15174 


16.3 kb 



Amplicon 
Primer Sequence Positions Size 

Right-hand primer for the human (5-globin gene cluster: 
RH1053 5'-GCACTGGCTTAGGAGTTGGACT-3' 

Left-hand primer for the human [5-globin gene cluster: 
RH1022 5'-CGAGTAAGAGACCATTGTGGCAG -3' 
RH1024 5'-TTGAGACGCATGAGACGTGCAG-3' 

Right-hand primer for the human mitochondrial DNA genome: 
RH 1 066 5'-TTTC ATCATGCGGAGATGTTGGATGG-3 ' 

Left-hand primer for the human mitochondrial DNA genome: 
RH 1 065 5'-TGAGGCCAAATATCATTCTGAGGGGC-3' 



misincorporated nucleotides. The XL PCR amplification protocol uses relatively short 
denaturation times at moderately high temperatures to minimize template damage while 
ensuring complete denaturation. The XL PCR protocol also uses a wax-mediated hot- 
start method, relatively high-annealing temperatures, and reduced enzyme levels to 
enhance reaction specificity. 

10. There are a variety of different enzyme and buffer systems for long PCR that are commer- 
cially available, as shown in Table 4. 

11. Ethidium bromide fluorescence is highly specific for double-stranded DNA. Prepare a 
standard curve using solutions of 10-400 ng of X/Hindlll DNA in 1.2 mL volume of 
0.5 (ig/mL ethidium bromide, 20 mM KH 2 P0 4 , and 0.5 mM EDTA (pH 11.8-12.0). The 
fluorescence of aliquots of template DNA can then be compared against these standards. 
The high pH is critical to minimize any RNA contribution to the fluorescence. An A-4 
Filter Fluorometer (Optical Technology Devices, Elmsford, NY) can be used with a 
bandpass filter (360 nm maximum) for excitation and an interference glass filter at 
610 nm for emission spectra (B. Van Houten, University of Texas Medical Branch, 
Galveston, TX, communication with S. Cheng). 

12. Higher molecular weight material will be better resolved on a 3% agarose gel than on a 
higher percentage gel. Because a 0.3% gel is fragile, use a high-strength agarose, such as 
Seakem Gold. Chill the gel at 4 C C before removing the comb. Submerge the gel in chilled 
buffer before removing the combs, as the wells may collapse. 

13. A few helpful Web addresses that contain tips on primer design, software to design prim- 
ers, and databases of DNA sequences are: 

http://www.chemie.uni-marburg.de/~becker/prim-gen.html (PrimerDesign); http://www. 
hybsimulator.com/design.html (HYB simulator); http://www.premierbiosoft.com/ 
primerdesign (Primer Premier); 

http://alces.med.umn.edu/VGC.html (Primer Selection). 

http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html (a collection of sites). 
Note that these addresses were found during a routine search (March 2001) of the Web 
and have not been evaluated or verified by the author. 



Table 4 

Some Commercially Available Long PCR Products 



Company 



Product 



Components 



Enzyme Souces 



For Use With 



Roche 
www.roche.com 



Expand High Fidelity 
PCR System 

Expand Long Template 
PCR Systems 

Expand 20 kb PCR System 



Taq DNA Polymerase 

Pwo DNA Polymerase 

10X Buffer, MgCL, 

Taq DNA Polymerase 

Pwo DNA Polymerase 

10X Buffers 

Taq DNA Polymerase 

Pwo DNA Polymerase 

10X Reaction Buffer 

MgCL, Buffer Solution 



CLONTECH 
www.clontech.com 

4\ 


Advantage cDNA 
Polymerase Mix and 
PCR Kit 


KlenTaq polymerase 

Tth Polymerase 

TaqStart antibody 

(Kit includes Buffer, dNTPs, 


CT> 




template, primer) 




Advantage Genomic 
Polymerase Mix and 
PCR Kit 


Tth polymerase 

Proofreading enzyme 

TaqStart antibody 

(Kit includes Buffer, dNTPs, 

template, primer) 


Strategene 

w w w . strategene .com 


TaqPlus Long PCR System 


TaqPlus Long Polymerase 

10X TaqPlus Long Reaction 

Buffer 


TaKaRa 

www.takara.com 


TaKaRa Ex Taq DNA 


Ex Taq polymerase 
10X Taq Buffer 
dNTP Mixture 




LA PCR Kit 


LA Taq polymerase 



10X LA PCR Buffer II 

Control K Template 

Primers, Markers 



Thermits aquaticus 
Pyrococcus woesel 

Thermits aquaticus 
Pyrococcus woesel 

Thermits aquaticus 
Pyrococcus woesel 



Thermus aquaticus 
Thermus thermophilics 



Thermus thermophilics 



Thermits aquaticus 
Pyrococcus furiosus 

Taq polymerase 
Proofreading enzyme 

Taq polymerase 
Proofreading enzyme 



<5 kb genomic DNA 



=26 kb genomic DNA 



=35 kb genomic DNA 



Up to 10 kb cDNA 



Up to 30 kb genomic DNA 



Up to 35 kb 



Up to 17 kb genomic DNA 



Up to 27 kb genomic DNA 



XL PCR Amplification of Long Targets 47 

14. For a manual hot start, assemble a single master mix comprised of all but one key reaction 
component (usually DNA polymerase, Mg(OAc)2, or dNTPs). Add a 75-80°C Hold (e.g., 
5 min for up to 20-25 samples) before the initial denaturation step of the thermal cycling 
profile (see Step 4 in Section 3.4.). Bring all samples to this hold temperature for approx 
1 min in the thermal cycler, then add the remaining component to each reaction mix. Note 
that the volume of this addition must be large enough to minimize pipeting variations, yet 
small enough to minimize the cooling effect on the reaction mixture already within the 
tube. If the rTth XL DNA polymerase or dNTP blend is the component that is withheld, 
dilute the components with IX XL Buffer II to facilitate complete mixing. Remember, to 
change pipet tips after each component addition to the reaction mix. 

15. XL PCR appears to be more sensitive to the integrity of the dNTP solutions than is stan- 
dard PCR. Stock dNTP solutions should be at pH 7.0-7.5. Reproducibility may be best if 
these solutions are aliquoted and subjected to a minimal number of freeze-thaw cycles. 
Changing dNTP stock solutions in an optimized PCR may require slight adjustment of 
the Mg(OAc) 2 concentration. 

16. XL PCR amplifications can be quite sensitive to Mg(OAc) 2 levels, and each new target or 
primer pair may have a different optimal range for the Mg(OAc) 2 concentration. In gen- 
eral, the acceptable Mg(OAc) 2 window empirically narrows as the starting DNA copy 
number is decreased and/or the amplicon length is increased. For optimal levels, titrate 
the Mg(OAc) 2 in increments of 0.1 mM. 

17. Repeated freeze-thawing of primers may reduce the efficiency of XL PCR. Make small 
working stock aliquots of primers and dispose after two or three freeze-thaw cycles. 

18. Tap or rotate the tube of AmpliWax PCR Gem 100s to empty a few beads onto either a 
clean sheet of weighing paper, into a clean weighing boat, or into the tube cap. Use a clean 
pipet tip to carefully direct a single bead into each tube containing the lower reagent mix. 

19. The optimal amount of rTth DNA polymerase, XL depends on target length and starting 
copies. For example, with the Perkin Elmer GeneAmp PCR System 9600, amplification 
of genomic targets at IX 10 4 starting copies requires two enzyme units of rTthXL/ per 
100 uL reaction, while amplification of Lambda DNA at 1X10 7 starting copies requires 
four enzyme units of rTthXL/100 uL reaction. Optimal enzyme concentration can be 
determined empirically by a titration of rTth DNA Polymerase, XL in increments of 
1 U/100 (iL reaction. 

20. In general, 50-100 ng of total human genomic DNA (with high-average single-stranded 
molecular weight) will suffice for a 100-(iL reaction. Excessive amounts of genomic DNA 
may contribute to the accumulation of nonspecific products. 

21. If the template volume represents a large fraction of the final reaction volume, the DNA 
should be diluted in water or 10 mM Tris-HCl, 0.1 mM EDTA, to minimize chelation of 
the Mg(OAc), in the final reaction mix (see Note 15). 

22. Long PCR amplifications may be more sensitive to potential reaction inhibitors than 
shorter target amplifications. In such cases, the addition of 50-500 ng/(xL nonacetylated 
BSA may enhance yields, possibly by binding nonspecific inhibitors. 

23. Amplification of GC-rich nucleic acids can often be problematic. Reagents including 
DMSO, glycerol, TMAC, betaine, and 7-deaza GTP have routinely been used to disrupt 
base pairing or isostabilize the DNA allowing efficient amplification of difficult templates 
(see Chapter 1). A combination of Betaine and DMSO, in particular, has recently been 
shown to suggest improved processivity. The mixture may increase the resistance of the 
polymerase to denaturation (25). 



48 Kolmodin 

24. Recently, Escherichia coli exonuclease III has been shown to be helpful for XL PCR 
amplifications using DNA samples induced with strand breaks and/or apurinic/ 
apyrimidinic sites via in vitro treatments such as high temperature (99°C), depurination at 
low pH and near-UV radiation. Exonuclease III also permitted amplification with DNA 
aged samples isolated by the phenol/chloroform method (26). 

25. XL PCR amplifications are sensitive to the times and temperatures of denaturation and 
annealing, thus different types of thermal cylcers are likely to require adjustments to the 
recommended thermal cycling profile. Profiles for all the Perkin-Elmer GeneAmp PCR 
instruments are provided with the GeneAmp XL PCR Kit. 

26. Complete denaturation of the template strands is critical for successful PCR amplifica- 
tions. The presence of extended GC-rich regions may require use of 95-96°C denatur- 
ation temperatures, but the time should be kept short to minimize damage to the 
single-stranded template and loss of rTth DNA polymerase, XL activity over the course 
of the PCR run. 

27. The choice of annealing temperature should be based on the actual primer pair being used 
(see Subheading 3.3.)- In general, the highest possible temperature should be used to 
minimize annealing to secondary priming sites. For reactions in which only the desired 
product is obtained, a lower annealing temperature may improve product yields. 

28. This thermal cycling profile uses two temperatures, compared to the three temperatures 
typically used in standard PCR. Strand synthesis by rTth DNA Polymerase, XL is efficient 
between 60-70 c C. Consequently, when an annealing temperature of approx 62-70°C is 
used, the same temperature can be set for the extension phase of the cycle. If lower 
annealing temperatures are necessary, a third temperature step at 65-70°C should be 
added for efficient completion of strand synthesis, but sufficient time at the annealing 
temperature must be allowed for efficient priming before the reactions are raised to the 
extension temperature. 

29. In general, use extension times sufficient for 30-60 s/kb of the target. In the two-tempera- 
ture cycling profile, this applies to the total annealing-plus-extension time. As product 
accumulates, the ratio of template to polymerase molecules will increase, and the overall 
reaction efficiency may decrease. The AUTO feature of the Perkin Elmer GeneAmp PCR 
System 9600 allows the extension time to be incrementally increased during late cycles 
of the run, which helps maintain reaction efficiency. The potential disadvantage of using 
very long extension times initially is that during early cycles, excessively long extension 
times may permit nonspecific products to accumulate. 

30. The optimal number of cycles will depend on the initial copy number of the template and 
the reaction efficiency. Reaction efficiency is generally higher for shorter (5-10 kb) vs 
longer (20-30 kb) targets. For example, from approx 104 copies of human genomic DNA 
(37 ng, in a 50-[iL reaction), the 16.3-kb multicopy mitochondrial genome target can be 
readily amplified with a total of 30-35 cycles, whereas the 17.7-kb single-copy (5-globin 
target requires at least 35-37 cycles. The number of AUTO cycles (9600) will also depend 
on the initial copy number of the template and the length of the target. 

31. Amplified target bands may be identified by size (gel mobility relative to standards), a 
Southern blot analysis (as in ref. 13), and/or analytical restriction digests. High-molecu- 
lar-weight smears tend to reflect high levels of nonspecific synthesis, as in the cases of 
excess rTth DNA polymerase, XL or excess Mg(OAc) 2 . Excessively long extension times 
or too many cycles of amplification can also result in the appearance of high-molecular 
weight bands or nonspecific smears. Low-molecular weight secondary bands may reflect 
insufficient specificity, and may be reduced by the use of a higher annealing temperature, 



XL PCR Amplification of Long Targets 49 

and/or lower concentrations of template, primers, or rTth DNA polymerase, XL. If accu- 
mulation of products other than the desired product is significant, the best solution may 
be to redesign one or both primers. Absence of any detectable product from a known 
template may indicate that the denaturation temperature was either too low for the tem- 
plate or too high for the DNA polymerase, XL; that the annealing temperature was too 
high for the primer pair; or that either the polymerase or the Mg(OAc) 2 concentration was 
too low. If a Southern blot analysis or reamplification using primers located within the 
original target (nested PCR) reveals that the desired product is present at a very low level, 
the explanation may be that too few cycles of amplification were used for the starting 
target copy number (see Notes 20 and 21). 

32. Optimization of the denaturation or annealing temperatures should be made in incre- 
ments of 1-2°C. Adjustments to enzyme concentration can be made in increments of 
0.5-1 U/100 \ih reaction. Optimization of the Mg(OAc^ concentration should be car- 
ried out in increments of 0.1 rr\M (see Notes 27 and 28). 

33. Carryover contamination (see Chapter 1) and dNTP stock solutions of poor quality can 
both significantly reduce the apparent optimal range for the Mg(OAc) 2 concentration. 
These problems may not be observed initially, but may become apparent during late 
amplifications with targets and primers previously observed to work well. 

34. Resolution of high-molecular weight DNA (>50 kb) is best achieved using pulse field, for 
example, field inversion electrophoresis (27). One such system is made by Hoefer (San 
Francisco, CA). 

35. The 3' ends of the PCR may have an additional one or two nontemplated nucleotides 
(28,29). Although the 3'-5' exonuclease activity present in rTth DNA Polymerase, XL 
would be expected to remove these 3'-additions, there is evidence that a certain fraction 
of XL PCR product molecules have an additional 3'-A (30). This fraction is likely to be 
less than that observed in standard PCR with Tag DNA polymerase, and using methods, 
such as the TA Cloning Kit (Invitrogen, San Diego, CA), which take advantage of the 3 A 
addition may therefore be inefficient. If necessary, the 3'-additions can be removed by 
incubation with Pfu DNA polymerse (31) or with Klenow fragment of E. coli DNA 
polymerse I (24,29). 

References 

1. Cheng, S., Chen, Y., Monforte, J. A., Higuchi, R., and Van Houten, B. (1995) Template 
integrity is essential for PCR amplification of 20- to 30-kb sequences from genomic DNA. 
PCR Meth. Appli. 4, 294-298. 

2. Cheng, S. (1995) Longer PCR amplifications, in PCR Strategies (Innis, M. A., Gelfand, 
D. H., and Sninsky, J. J., eds.) Academic, San Diego, CA, pp. 313-324. 

3. Cheng, S., Chang, S.-Y., Gravitt, P., and Respess, R. (1994) Long PCR. Nature 369, 
684-685. 

4. Cheng, S., Fockler, C, Barnes, W. M., and Higuchi, R. (1994) Effective amplification of 
long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. USA 91, 
5695-5699. 

5. Barnes, W. M. (1994) PCR amplification of up to 35 kb with high fidelity and high yield 
from X bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216-2220. 

6. Brugnoni, R., Morandi, L., Brambati, B., Briscioli, V., Cornelio, F., and Mantegazza, R. 
(1998) A new non-radioactive method for the screening and prenatal diagnosi of myotonic 
dystrophy patients. J. Neurol. 245, 289-293. 



50 Kolmodin 

7. Salminen, M. O., Koch, C, Sanders-Buell, E„ Ehrenberg, P. K., Michael, N. L., Carr, J. 
K., Burke, D. S., and McCutchan, F. E. (1995) Recovery of virtrually full length HIV-1 
provirus of Diverse Subtypes from primary virus cultures using the polymerase chain reac- 
tion. Virology 213, 80-86. 

8. Van Houten, B., Cheng, S., and Chen, Y. (2000) Measuring gene-specific nucleotide exci- 
sion repair in human cells using quantitative amplification of long targets from nonogram 
quantities of DNA. Mutation Research 460, 81-94. 

9. Landre, P. A., Gelfand, D. H., and Watson, R. M. (1995) The use of cosolvents to enhance 
amplification by the polymerase chain reaction, in PCR Strategies (Innis, M. A., Gelfand, 
D. H., and Sninsky, J. J., eds.) Academic, San Diego, CA, pp. 3-16. 

10. Skera, A. (1992) Phosphorothioate primers improve the amplification of DNA sequences 
by DNA polymerase with proofreading activity. Nucl. Acids Res. 20, 3551-3554. 

1 1. de Noronha, C. and Mullins, J. (1992) PCR Meth. Appli. 2, 131-136. 

12. Miller, S. A., Dykes, D. D., and Polesky, H. F. (1988) A simple salting out procedure for 
extracting DNA from human nucleated cells. Nucl. Acids Res. 16, 1215. 

13. Sambrook, J., Fitsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory 
Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 6.20, 
6.21, 9.16-9.19, 9.34-9.57, and B.23-B.24. 

14. Innis, M. A. and Gelfand, D. H. (1990) Optimization of PCRs, in PCR Protocols (Innis, 
M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) Academic, San Diego, CA, pp. 
3-12. 

15. Wu, D. Y., Ugozzoli, L., Pal, B. K., Qian, J., and Wallace, R. B. (1991) The effect of 
temperature and oligonucleotide primer length on the specificaity and efficiency of ampli- 
fication by the polymerase chain reaction. DNA Cell Biol. 10, 233-238. 

16. Schmid, C. W. and Jelinek, W. R. (1982) The Alu family of dispersed repetitive sequences. 
Sciences 216, 1065-1070. 

17. Chou, Q., Russell, M., Birch, D. E., Raymond, J., and Block, W. (1992) Prevention of pre- 
PCR mis-priming and primer dimerization improves low-copy-number amplifications. 
Nucl. Acids Res. 20, 1717-1723. 

18. Monforte, J. A., Winegar, R. A., andRudd, C.J. (1994) Megabase genomic DNA isolation 
procedure for use in transgenic mutagenesis assays. Environ. Mol. Mutagen. 23, 46. 

19. Mullenbach, R., Lagoda, P. J. L., and Welter, C. (1989) Technical Tips: an efficient salt- 
chloroform extraction of DNA from blood and tissues. Trends in Gen. 5, 391. 

20. Kolmodin, L., Cheng, S., and Akers, J. (1995) GeneAmp XL PCR Kit, in Amplifications: 
A Forum for PCR Users (The Perkin Elmer Corporation), Issue 13. 

21. Cheng, S., Higuchi, R., and Stoneking, M. (1994) Complete mitochondrial genome ampli- 
fication. Nature Gen. 7, 350, 351. 

22. Robin, E. D. and Wong, R. (1988) Mitochondrial DNA molecules and virtual number of 
mitochondria per cell in mammalian cells. ./. Cell Physiol. 136, 507-513. 

23. Jurecic, R., Nachtman, R. G., Colicos, S. M., and Belmont, J. W. (1998) Identification and 
Cloning of Differentially Expressed Genes by Long-Distance Differential Display. Anal. 
Biochem. 259, 235-244. 

24. Scharf, S. J., (1990) Cloning with PCR, in PCR Protocols (Innis, M. A., Gelfand, D. H., 
Sninsky, J. J., and White, T. J., eds.) Academic, San Diego, CA, pp. 84-91. 

25. Fromenty, B., Demeilliers, Mansouri, A., and Pessayre, D. (2000) Escherichia coli exonu- 
clease III enhances long PCR amplification of damaged DNA templates. Nucl. Acids Res. 
28, 50. 



XL PCR Amplification of Long Targets 51 

26. Carle, G. F., Frank, M., and Olson, M. V. (1989) Electrophoretic separations of large 
DNA molecules by periodic inversion of the electric field. Science 232, 65-68. 

27. Hengen, P. N. (1997) Methods and reagents: optimizing multiplex and LA-PCR with 
betaine. TIBS 22, 225,226. 

28. Clark, J. M. (1988) Novel nontemplated nucleotide addition reactions catalyzed by 
prokaryotic and eucaryotic DNA polymerases. Nucl. Acids Res. 16, 9677-9686. 

29. Hu, G. (1993) DNA Polymerase-catalyzed addition of nontemplated extra nucleotides to 
the 3' end of a DNA fragment. DNA Cell Biol. 12, 763-770. 

30. Stewart, A. C, Gravitt, P. E., Cheng, S., and Wheeler, C. M. (1995) Generation of entire 
human papilloma virus genomes by long PCR: frequency of errors produced during ampli- 
fication. Genome Res. 5, 79-88. 

31. Costa, G. L. and Weiner, M. P. (1994) Protocols for cloning and analysis of blunt-ended 
PCR-generated DNA fragments. PCR Meth. Appl. 3, S95-S106. 



Coupled One-Step Reverse Transcription 
and Polymerase Chain Reaction Procedure 
for Cloning Large cDNA Fragments 

Jyrki T. Aatsinki 
1. Introduction 

Although Thermus aquaticus (Taq) and Thermus thermophilus (Tth) DNA poly- 
merases have the ability to reverse transcribe RNA to complementary DNA (cDNA) 
and subsequently amplify the target cDNA, they are not usually the first choices for 
reverse transcription-polymerase chain reactions (RT-PCR) (1-4). Because they only 
synthesize short cDNA fragments, their use is not widespread. In general, avian myelo- 
blastosis virus (AMV), or moloney murine leukemia virus (M-MLV) reverse tran- 
scriptases (RTs) are used to reverse transcribe RNA to cDNA templates for subsequent 
PCR. Previous coupled methods are also unable to amplify large cDNA fragments 
and, thus, they are suitable only for the detection of gene expression (5-8). The one- 
step RT-PCR procedure presented here was developed to amplify large cDNA frag- 
ments suitable for cloning full-length open reading frames (ORFs) encoding rat LH/ 
CG receptor isoforms (9-12). As we all know, the construction of clones, including 
library screening and restriction mapping, by conventional cloning methods is very 
laborious and difficult. 

The one-step RT-PCR procedure was first optimized for its specificity. Low con- 
centrations of dNTP (0.2 mM of each), MgCl 2 (1.5 mM), and primer (0.1 (xM of each) 
and a relatively high annealing temperature (55°C) were used, because these condi- 
tions have been found to enhance specific amplification. The commercially available 
PCR buffer (10 mM Tris-HCl, pH 8.4, and 50 mM KC1) was found to be suitable for 
primer extension by AMV-RT although it differed in its constituents from the recom- 
mendations of the manufacturer. To assure that primer extension was completed, long 
extension times were used, both in reverse transcription and PCR (60 min and 10 min 
+ 59 s/cycle, respectively). Possible aggregates and secondary structures were elimi- 
nated by denaturing both primers and RNA at 65°C, for 15 min, before starting the 
amplification. Subsequent to a 1 h incubation at 42°C, the temperature was raised to 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

53 



54 Aatsinki 

95°C for 3 min to dissociate RNA-cDNA hybrids. Finally, RT-PCR products could be 
easily cloned for in vitro translation studies and for transfections in different cell lines, 
because suitable restriction enzyme sites were incorporated at both ends. Examples of 
other potential applications of the present coupled RT-PCR procedure, in addition to 
cDNA cloning and the detection of gene expression, include the quantitation of mRNA 
and clinical diagnostics (e.g., the detection of viral RNA, tumor cells, parasites, and 
genetic disorders). Our procedure has been used for over a decade and it still meets 
current demands. Although commercial applications have become available, the author 
thinks that the coupled one-step RT-PCR procedure was the first and still the best 
available procedure to produce large RT-PCR products sensitively and reliably. 

2. Materials 

2. 1. Coupled One-Step RT-PCR 

1 . Total RNA isolated by the TRIZOL® following the instructions of manufacture (Gibco-BRL, 
Gaithersburg, MD). 

2. Oligonucleotide primers, typically 32-35 nucleotides long and with a 40-60% G + C 
content, are designed to have internal unique restriction sites and an additional 8-9 nucle- 
otide complementary sequence at the 5' end of the restriction sites. These added nucle- 
otides are important for helping restriction enzymes to cleave the RT-PCR product. When 
creating a new restriction site, select a sequence that requires as few changes as possible. 
Primer-dimer formation during PCR is best avoided by using primers having 
noncomplementary 3' ends. 

3. Ribonuclease inhibitor, Inhibit-ACE (5 prime-3 prime, Boulder, CO). 

4. 10X PCR buffer: 500 mM KC1, 100 mM Tris-HCl, pH 8.4 (Gibco-BRL). 

5. 50 mM MgCl 2 (Gibco-BRL). 

6. Deoxynucleotide triphosphates (dNTPs) (Pharmacia, Uppsala, Sweden). 

7. AMV-RT (Promega, Madison, MI). 

8. Tctq DNA polymerase, recombinant (Gibco-BRL). 

9. DNA Engine™ Peltier Thermal Cycler (MJ Research, Inc., Watertown, MA). 

10. 200 uL-fhin-wall PCR tubes. 

11. 6X loading dye solution (MBI Fermentas, Vilnius, Lithuania). 

12. DNA electrophoresis size markers: GeneRuler™ Ladder Plus, ready-to-use (MBI 
Fermentas). 

13. Reagents and supplies for agarose gel electrophoresis: See preparation of mixtures and 
use of equipments in the laboratory manual (13). 

2.2. Cloning of RT-PCR Product 

1. Wizard PCR product purification column (Promega). 

2. Restriction enzymes (e.g., BamHl and EcoRI) (Pharmacia). 

3. Plasmid DNA, pUCBM20 (Boehringer Mannheim GmbH, Mannheim, Germany) 

4. Escherichia coli (E. Coli) IM109 strain. 

5. T4 DNA ligase (high concentration; 5 U/uL) and supplied 10X ligase buffer (Boehringer 
Mannheim GmbH). 

6. Alkaline phosphatase and supplied 10X alkaline phosphatase buffer (Promega). 

7. Reagents for DNA precipitation: 4 M NaCl; absolute ethanol; 70% ethanol. 

8. Reagents and supplies for molecular cloning: 1 M CaCl 2 ; isopropyl-(5-D-thio-galactoside 
(IPTG); 5-bromo-4-chloro-3-indolyl-|3-D-galactopyranoside (X-gal); Luria-Bertani medium; 
Luria-Bertani medium plates. See preparation of mixtures in the laboratory manual (13). 



One-Step RT-PCR Procedure 55 

3. Methods 

3. 1. Coupled One-Step RT-PCR 

1 . Dilute primers to a concentration of 20 ng/(xL in sterile distilled H 2 0. Add 2.5 \iL of each 
primer to a reaction tube containing a volume of sterile distilled H 2 sufficient to bring 
the total reaction volume to 50 uL after the addition of the rest of the reactants from steps 
3 and 4. Add 0.5 U Inhibit-ACE and incubate for 20-30 min at room temperature. 

2. Prepare stock mixtures of 10X PCR buffer (containing a final concentration of 1.5 vaM 
MgCl 2 in IX PCR buffer) and 10 mM dNTPs (2.5 mM of each dNTP) and incubate for 
20-30 min at room temperature after adding 1 U Inhibit-ACE/ 100 uE of stock mixture. 

3. Add total RNA (100 pg-10 u.g) to the reaction tube (see step 1), denature at 65°C for 15 min 
and cool to 4°C using a programmed DNA thermal cycler (see Note 1). 

4. Add 6.5 uL of 10X PCR buffer and 4 uL of 10 mM dNTPs from step 2 to the reaction 
tube. Add 10 U AMV-RT and 2.5 U DNA polymerase, mix carefully, and collect by brief 
centrifugation. 

5. Incubate at 42°C for 1 h to allow RT. 

6. Step 5 is linked to PCR cycles. Initial denaturation at 95°C for 3 min, followed by 30 
cycles consisting of denaturation at 95°C for 1 min, primer annealing at 55 C C for 2 min, 
and extension at 72°C for 10 min + 59 s/cycle. 

7. Take 5-25 uE of the RT-PCR product, add gel loading buffer, and size fractionate on a 

I % ethidium bromide-stained agarose gel. Gel electrophoresis of the RT-PCR products 
are shown in Fig. 1 (see Notes 2-6). 

3.2. Multiplex Coupled One-Step RT-PCR 

1. Primers for low-copy number mRNA are added as aforementioned in Subheading 3.1., 
step 1, where 50 ng of each of the LH/CG receptor primer was added to a reaction tube. 
Primers for more abundant mRNAs are reduced such as 25 ng of both carbonic anhydrase 

II primers and 1 ng of both B-actin primers are sufficient to produce visible bands on the 
ethidium bromide-stained agarose gel (see Fig. 1, lane 5). Mix all the primers to a reac- 
tion tube containing a volume of sterile distilled H 2 sufficient to bring the total reaction 
volume to 50 uL after the addition of the rest of the reactants from steps 3 and 4. Add 0.5 U 
Inhibit-ACE and incubate for 20-30 min at room temperature (see Note 7). 

2. Follow step 2 from Subheading 3.1. 

3. Follow step 3 from Subheading 3.1. 

4. Add 6.5 uL of 10X PCR buffer and 4 uL of 10 mM dNTPs from step 2 to the reaction 
tube. Add 20 U AMV-RT and 2.5 U DNA polymerase, mix carefully, and collect by brief 
centrifugation. 

5. Follow steps 5-7 from Subheading 3.1. 

3.3. Cloning of RT-PCR Product 

1. Purify the RT-PCR product using a Wizard PCR product purification column following 
the manufacturer's instructions. 

2. Digest the purified RT-PCR product with restriction enzymes using a several-fold excess 
of enzyme and long incubation times. Usually, 50 U of restriction enzyme can be added at 
the beginning of digestion and a further 20 U during the incubation, which can be done 
overnight (see Note 8). 

3. Purify the sample as in step 1. 

4. Precipitate the sample by adding 1/20 vol of 4 M NaCl and 2 vol of cold absolute ethanol, 
allow to stand overnight at -20°C, or for 30 min at -80°C. Centrifuge at 12 OOOg for 



56 



Aatsinki 



1 2 3 4 5 6 

3000 ^^^^^M ' 3000 




Fig. 1. Coupled one-step RT-PCR amplification of three different cDNA species from rat 
ovary total RNA. Lane 2 shows LH/CG receptor isoforms amplified using CAATT 
TTGGAATTCTAGTGAGTTAACGCTCTCG as the reverse primer, GGGAGCTCGA 
ATTCAGGCTGGCGGGCCATGGGGCGG as the forward primer (mismatched nucleotides, 
to create internal restriction sites, are underlined), and 5 (xg of total RNA as the template. Lane 
3 shows the carbonic anhydrase II RT-PCR product amplified using GAGCACTATCCAG 
GTCACACATTCCAG as the reverse primer, ACTGGCACAAGGAGTTCCCCATTGCCA as 
a forward primer, and 100 ng of total RNA as the template. Lane 4 shows the (3-actin RT-PCR 
product amplified using GATGCCACAGAATTCCATACCCAGGAAGGAAGGC as the 
reverse primer, GCCGCCCTAGGATCCAGGGTGTGATGGTGGGTAT as the forward primer 
(mismatched nucleotides, to create internal restriction sites, are underlined), and 100 pg of total 
RNA as the template. Lane 5 shows the multiplex RT-PCR, where three different cDNA spe- 
cies were simultaneously amplified in same reaction tube using the above mentioned primers, 
and 5 (xg of total RNA as the template. Other conditions for multiplex RT-PCR are as men- 
tioned in Subheading 3.2. 25 uL of RT-PCR products were size-fractionated on a 1% ethidium 
bromide- stained agarose gel. Lanes 1 and 6 show 10 |xL of the molecular weight standards 
(GeneRuler™ Ladder Plus). 



20 min at +4 C C and discard the supernatant. Add 1 mL of 70% ethanol to the pellet and 
centrifuge as before. Dissolve the dried pellet in 20 uL of sterile distilled H 2 0. 

5. Prepare plasmid DNA by digesting with suitable restriction enzymes for 2-3 h using 
10 U/l u.g DNA. Dephosphorylate the DNA according to the manufacturer's instructions 
if using a single restriction enzyme. Purify the sample as described in steps 1 and 4. 

6. Set up three ligation mixtures using 3:1, 1:1, and 1:3 molar ratios of insert and plasmid 
DNA. Add sterile distilled H-,0 to final volume of 8 uL, heat at 45 °C for 5 min and cool 
to 16°C. Add 1 fiL of 10X ligation buffer, 1 uL of high concentration T4 DNA ligase 
(5 U/uL), and incubate overnight at 16°C. 

7. Transform competent cells using standard laboratory protocols (13). Pick up positive 
clones and analyze them by restriction digestion and agarose gel electrophoresis. Deter- 
mine the nucleotide sequences of some clones by the double-stranded dideoxy sequenc- 
ing method (14), 



One-Step RT-PCR Procedure 57 

4. Notes 

1. It is always necessary to test the amount of RNA for optimal amplification. If the total 
RNA contains high amounts of the target mRNA, efficient amplification is obtained with 
picogram amounts of RNA. On the other hand, if the total RNA contains only very small 
amounts of the target mRNA, up to 10 u,g of the total RNA can be used to obtain an 
efficient amplification. 

2. If no specific bands are visible on the ethidium bromide-stained agarose gel, use a gradu- 
ally increasing amount of reverse transcriptase. Do not use excess amounts of Taq DNA 
polymerase, because this has been reported to lower the amount of specific RT-PCR prod- 
uct (10,12). 

3. If no specific bands are visible on the ethidium bromide-stained agarose gel after optimi- 
zation, prepare a Southern blot (15) of the gel. Hybridize with an appropriate probe that 
does not contain overlapping sequences with the primers used for the RT-PCR. 

4. If, after Southern blotting, a specific hybridization signal is obtained, use nested PCR to 
produce visible bands on the ethidium bromide-stained agarose gel. Prepare a new pair of 
primers located further outside the region where the first primer set was designed. This 
new primer set does not need modifications (e.g., restriction sites) and can be shorter 
(about 22-25 nucleotides). Use these new primers in RT-PCR; take 1-5 uL of the 
RT-PCR product and use it as a template and the original modified oligonucleotides as 
primers for the second round of PCR. 

5. If no specific bands are seen after procedures described in Notes 1-4, check the total 
RNA used in the experiments. Use primers of abundant mRNA (e.g., (J-actin), instead of 
your primers, in the coupled one-step RT-PCR procedure. A positive signal in the control 
reaction leaves only two possibilities for explaining negative results: the sample RNA 
does not contain the target template RNA, or the primers anneal inefficiently to the tem- 
plate RNA. Try a new pair of primers located in a different region of the cDN A, because 
primers are sometimes chosen in a region of secondary structure, causing difficulties in 
priming. 

6. Negative controls in RT-PCR should be done to eliminate the possibility of potential 
DNA contamination. Prepare two control samples following the above procedure, but 
omit the template RNA in one control and omit RT in the second. 

7. Test empirically the amount of primers used in multiplex coupled one-step RT-PCR. 
Primers for low-copy number mRNAs are added as in basic procedure and primers for 
more abundant mRNAs are reduced until all the RT-PCR products are visible on the 
ethidium bromide-stained agarose gel. Excess amount of primers for abundant mRNAs 
compete efficiently for enzymes resulting no visible RT-PCR product of rare mRNAs. 

8. The procedure for directional cloning of RT-PCR products is also included in this chapter 
because it has been problematic for many laboratories. In the present procedure, the use 
of a several-fold excess of restriction enzymes and long incubation times are critical for 
optimal results. Commercial methods for cloning PCR products (e.g., T/A cloning and 
blunt end DNA ligation kits) are also recommended, although they are expensive to use. 

Acknowledgments 

This work was performed at the Department of Anatomy and Cell Biology, and the 
Institute of Dentistry, University of Oulu, Finland. The support of grants from the 
Finnish Cultural Foundation and the Memorial Foundation of Maud Kuistila are grate- 
fully acknowledged. 



58 Aatsinki 

References 

1. Jones, M. D. and Foulkes, N. S. (1989) Reverse transcription of mRNA by Thermits 
aquaticus DNA polymerase. Nucl. Acids Res. 17, 8387-8388. 

2. Shaffer, A. L., Wojnar, W., and Nelson, W. (1990) Amplification, detection, and auto- 
mated sequencing of gibbon interleukin-2 mRNA by Thermus aquaticus DNA polymerase 
reverse transcription and polymerase chain reaction. Analyt. Biochem. 190, 292-296. 

3. Tse, W. T. and Forget, B. G. (1990) Reverse transcription and direct amplification of 
cellular RNA transcripts by Taq polymerase. Gene 88, 293-296. 

4. Myers, T. W. and Gelfand, D. H. (1991) Reverse transcription and DNA amplification by 
a Thermus thermophilics DNA polymerase. Biochemistry 30, 7661-7666. 

5. Goblet, C., Prost, E., and Whalen, R. G. (1989) One-step amplification of transcripts in 
total RNA using the polymerase chain reaction. Nucl. Acids Res. 17, 2144. 

6. Singer-Sam, J., Robinson, M. O., Bellve, A. R., Simon, M. I., and Riggs, A. D. (1990) Meas- 
urement by quantitative PCR of changes in HPRT, PGK- 1 , PGK-2, APRT, MTase, and Zfy 
gene transcripts during mouse spermatogenesis. Nucl. Acids Res. 18, 1255-1259. 

7. Zafra, F., Hengerer, B., Leibrock. J., Thoenen, H., and Lindholm, D. (1990) Activity 
dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by 
non-NMDA glutamate receptors. EMBO J. 9, 3545-3550. 

8. Wang, R.-F., Cao, W.-W., and Johnson M. G. (1992) A simplified, single tube, single 
buffer system for RNA-PCR. BioTechniques 12, 702-704. 

9. Aatsinki, J. T., Pietila, E. M., Lakkakorpi, J. T., and Rajaniemi, H. J. (1992) Expression of 
the LH/CG receptor gene in rat ovarian tissue is regulated by an extensive alternative 
splicing of the primary transcript. Mol. Cell. Endocrinol. 84, 127-135. 

10. Aatsinki, J. T., Lakkakorpi, J. T., Pietila, E. M., and Rajaniemi, H. J. (1994) A coupled 
one-step reverse transcription PCR procedure for generation of full-length open reading 
frames. BioTechniques 16, 282-288. 

11. Aatsinki, J. T. (1997) Coupled one-step reverse transcription and polymerase chain reac- 
tion procedure for cloning large cDNA fragments, in Methods in Molecular Biology, vol. 
67, PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering (White, 
B.A., ed.) Humana, Totowa, NJ, pp. 55-60. 

12. Aatsinki, J. T., Lakkakorpi, J. T., Pietila, E. M., and Rajaniemi, H. J. (1998) A coupled 
one-step reverse transcription PCR procedure for generation of full-length open reading 
frames, in BioTechniques® Update Series, The PCR Technique: RT-PCR (Siebert, P., ed.), 
Eaton, Natick, MA, pp. 261-268. 

13. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory 
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

14. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chain-terminat- 
ing inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 

15. Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated 
by gel electrophoresis. ./. Mol. Biol. 98, 503-517. 



Long Distance Reverse-Transcription PCR 
Volker Thiel, Jens Herold, and Stuart G. Siddell 

1. Introduction 

Polymerase chain reaction (PCR) has been applied to the amplification of long 
DNA fragments from a variety of sources, including genomic, mitochondrial, and viral 
DNAs (1-5). We have adapted the concept of long PCR technology to reverse-tran- 
scription (RT) PCR (6). Here, we describe the parameters critical in producing 
RT-PCR products of up to 20 kbp. The nature of RT-PCR requires the synthesis of a 
cDNA by RT prior to its amplification in the PCR reaction. Thus, we focus on the 
three steps of RT-PCR: the preparation and requirements of the RNA template, the 
reverse transcription reaction, and the amplification of the cDNA by PCR. 

To carry out these studies, we used the genomic RNA of the human coronavirus 
HCoV 229E as template (7). The HCoV 229E genomic RNA has a length of ca. 27,000 
nucleotides and the homogeneity of the RNA can be readily assessed by electrophore- 
sis and hybridization analysis (8). HCoV 229E genomic RNA has two major advan- 
tages for the studies reported here. First, as a viral RNA, it is relatively abundant in the 
infected cell. Second, coronaviruses are positive strand RNA viruses and the genomic 
RNA has a 3' poly adenylate tract that can be used for affinity chromatography (9). 

In Subheading 3.1., we describe a simple and fast technique to purify poly(A)- 
containing RNA from tissue culture cells. In fact, we believe that the integrity and 
purity of the RNA template is the most critical parameter when the RT-PCR amplifi- 
cation of sequences more than 5 kb in length is desired (6) (see Note 4, Fig. 1). Depend- 
ing on the source of the RNA template, a method of preparation should be chosen that 
minimizes degradation of the RNA. In our hands oligo (dT)-based affinity chromatog- 
raphy with magnetic beads has proven to be reliable for the isolation of poly(A)-RNA 
that can be used to produce cDNA of more than 20 kb by RT. 

The conditions of the reverse transcription reaction strongly influence the outcome 
of the subsequent PCR. Reverse transcription reactions have been performed using the 
RNase H-deficient reverse transcriptase, Superscript II and the cDNA has been used 
for PCR amplification without further purification. In general, an RT-primer should 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

59 



60 Thiel, Herold, and Siddell 

tfp S I'l IS i> 23 

s. I J 1 1 " 

f op- MJfA 

BS 3j 



RHA.Z HKkb 




(27 



LI Jfcbp 



!47 


I^L~T klsp 


II 

-*- 


'£ 


|T.1|(tl(l 




| 


EWklr 


IL 


LSI! 


Z'l .' Idbp 


II 



RHA3 52tU g 1""P Jg 



RHA.1 i&W 
RNAS JjSI* 
KNAG Ukfc 

RNA7 1.7 Sbi 



Fig. 1. Northern hybridization analysis of HCoV 229E poly(A)-containing RNA isolated 
from infected MRC-5 cells. The material shown in lane 1 was prepared using poly(U)- 
Sepharose (6). The material shown in lane 2 was prepared using oligo(dT) 25 magnetic beads as 
described in Subheading 3.1. The poly(A)-containing RNAs were separated by gel electro- 
phoresis and the viral mRNAs were hybridized to a 32 P-(5'end)-labeled oligonucleotide 
(5AGAAACTTCTCACGCACTGG 3'). Also shown is the relationship of the HCoV 229E 
genomic RNA template, oligonucleotide primers and RT-PCR products. The oligonucleotides 
are indicated as arrows with their orientation and position relative to the HCoV 229E genomic 
RNA. The expected sizes of the RT-PCR products are indicated. 



be used that is highly specific and great care should be taken to adjust the optimal 
concentration of the RT-primer in the reverse transcription reaction. In our experi- 
ence, the major problem that arises is "less stringent" priming during the RT reaction. 
The fortuitous cDNAs that are synthesized and the small amounts of RT-primer that 
are carried over into the PCR are responsible for most of the background amplification 
products observed (6). 

Finally, the optimal conditions, regarding the amount of cDNA template, the choice 
of PCR primers and the cycle profile of the PCR, have to be determined. In this respect, 
the considerations that apply to the PCR amplification of dsDNA templates are equally 
applicable to amplification of cDNA produced by reverse transcription. 

2. Materials 

1. Phosphate-buffered saline (PBS). 

2. Lysis buffer: 10 ratf Tris-HCl, pH 7.5, 140 mM NaCl, 5 mM KC1, 1% Nonidet P-40 
(NP40). 

3. 01igo(dT) 25 -Dynabeads (Dynal) (3.3 x 10 8 beads/mL). 

4. Dynal Magnetic Particle Concentrator (Dynal). 



Long Distance RT-PCR 61 

5. 2X binding buffer: 20 mM Tris-HCl, pH 7.5, 1 M LiCl, 2 mM ethylenediamine tetraacetic 
acid (EDTA), 1% sodium dodecylsulfate (SDS). 

6. Wash buffer: 10 mM Tris-HCl, pH 7.5, 150 mM LiCl, 1 mMEDTA, 1 mM EDTA pH 7.5. 

7. Superscript II reverse transcriptase (Life Technologies). 

8. 5X first-strand buffer (Life Technologies). 

9. lOmMdNTPs(lOmMofeachdNTP). 

10. 0.1 M dithiothreitol (DTT). 

1 1 . RNasin (50 U/uL) (Pharmacia). 

12. Thin-wall PCR tubes. 

13. Elongase Enzyme Mix (Life Technologies). 

14. PCR buffer B (Life Technologies). 

3. Methods 

3.1. The RNA Template: Preparation of Poly(A)-Containing RNA 
Using Oligo(dT) 25 -Dynabeads 

The protocol below describes the isolation of poly(A)-containing RNA from a 
confluent layer of adherent cells grown in a 175 cm 2 tissue culture flask (see Note 1). 

1. Wash 200-500 uL oligo(dT) 25 -Dynabeads twice with 2X binding buffer using an 
appropriate Dynal Magnetic Particle Concentrator (see Note 2) and resuspend the beads 
in 1 .5 mL 2X binding buffer. 

2. Wash the cells twice with ice-cold PBS and then scrape in 10 mL ice-cold PBS. 

3. Pellet the cells at lOOOg for 2 min at 4°C. 

4. Resuspend the cell pellet in 1.5 mL ice-cold lysis buffer and incubate for 30 s on ice. 

5. Centrifuge the cell lysate at 1500g for 1 min at 4°C to remove nuclei. 

6. Mix the supernatant with oligo(dT) 25 -Dynabeads resuspended in 1.5 mL 2X binding 
buffer (Step 1) and incubate for 5 min at 23°C. Gently mix the sample every 1-2 min. 

7. Wash the oligo(dT) 25 magnetic beads twice with wash buffer using an appropriate Dynal 
Magnetic Particle Concentrator (see Note 3). 

8. Completely remove the wash buffer and add 50-100 [xL 2 mM EDTA, pH 7.5. 

9. Transfer the solution into a 1.5-mL Eppendorf tube and incubate for 2 min at 65°C to 
elute the bound poly(A)-RNA. 

10. Take the supernatant containing the poly(A)-RNA, add 5 uL RNasin and store at -70°C 
in aliquots (see Note 4). 

11. Regenerate the oligo(dT) 25 -Dynabeads according to the manufacturer's instructions 
(optional; see Note 5) 

3.2. The RT Reaction 

1. Add the following components to a volume of 19 uL (see Note 6): 
RNase-free water 
4 (xL 5X first strand buffer; 
2 uL 10 mM dNTPs (10 mM of each dNTP); 
2uL0.1MDTT; 
0.5 uL RNasin (50 U/uL); 

5-15 pmol reverse transcription primer (see Note 7); 

10-500 ng poly(A)-RNA (0.5-3 uL °f poly(A)-containing RNA prepared as 
described earlier). 



62 Thiel, Herold, and Siddell 

2. Incubate for 2 min at 42°C (see Note 8). 

3. Add 1 u,L (200 U) Superscript II reverse transcriptase. 

4. Incubate for 60-90 min at 42°C (see Note 9). 

5. Incubate for 2 min at 94°C and chill on ice (see Note 10). Store at -20°C until use. 

3.3. The PCR Reaction 

To amplify long DNA fragments from cDNA templates the basic principles of long 
PCR technology are applicable (see Note 11). The protocol below describes a typical 
reaction using the Elongase Enzyme Mix (Life Technologies). In addition, we provide 
a list of PCR cycle profiles (Fig. 2) and corresponding oligonucleotide primers 
(Fig. 1, Table 1) that have been used to produce RT-PCR products of 11.5-20.3 kbp 
in length. 

1. Set up the PCR reaction mix in thin-wall PCR tubes on ice [50 uL volume; final concen- 
trations: 60 mM Tris-S0 4 (pH 9.1), 18 mM (NH 4 ) 2 S0 4 , 2 mM MgS0 4 , 0.2 mM dNTPs, 
0.2-0.4 uM PCR primer]: 

10 uL buffer B (Life Technologies) (see Note 12); 

1 uL 10 mM dNTPs (10 mM of each dNTP); 

1-2 uL forward PCR primer (10-40 pmol); 

1-2 (xL reverse PCR primer (10-40 pmol); 

0.2-3 uL RT-reaction; 

1 uL Elongase Enzyme Mix (1 U/uL); 

water to a final volume of 50 uL; 

2. Place PCR tubes in an appropriate PCR cycler at 94°C (see Note 13). 

Cycle conditions: 

1 min 94°C, followed by 30 cycles of 20 s denaturation at 94°C, 20 s annealing at 
50°C and elongation for 1 min/kb expected product length at 68°C. Increase the elonga- 
tion time during the last 18 cycles by 15-30 s in each successive cycle. Incubate addi- 
tional 10 min at 72°C and terminate the reaction by decreasing the temperature to 4°C. 

4. Notes 

1. The number of cells will be about 10 7 -10 8 depending on the tissue culture cell line used. 
We recommend preparing the RNA from a confluent cell layer using at least one 175 cm 2 
tissue culture flask. This ensures that you will get enough poly(A)-containing RNA to 
perform several RT reactions using the same RNA preparation. 

2. There are different Magnetic Particle Concentrators recommended, depending on the size 
of the tubes. To lyse the cells and bind the RNA to the oligo(dT) 25 magnetic beads, we use 
15 mL tubes. Before we elute the poly(A)-containing RNA, we transfer the oligo(dT) 25 
magnetic beads into a 1.5-mL Eppendorf tube. 

3. If the oligo(dT) 25 magnetic beads appear to clump and do not resuspend well, you have 
cellular DNA in your sample. However, you can proceed without affecting the RNA qual- 
ity or yield, if you perform additional washing steps (2-4) until the oligo(dT) 25 magnetic 
beads can be resuspended easily. 

4. We strongly recommend analyzing the RNA template integrity before cDNA synthesis. 
We routinely perform a northern hybridization analysis. In Fig. 1 poly(A)-containing 
RNA prepared from HCoV 229E infected MRC-5 cells using two different methods are 
shown. In both RNA preparations, it is possible to identify the HCoV 229E genomic 
RNA (27.3 kb) and the six subgenomic mRNAs (1.7 kb-6.8 kb) that are characteristic of 



Long Distance RT-PCR 



63 



■£* ■& -^ ~$> ■£P Jp 1 



310 



3iJ 




KT TCJR prolan 



PL'RcjTjEppjI'il* 



ll.Skbf, 




1*A 



WT .Wkc 



Lfi.Tkbf. 
t73kbfi 



12)1 



16*. 



MT linin 
3Chci: 




|>er uycfci 



ffi-L 1 Mtaibi 



20.1 kbp 




25S 



3jnin( i 30 «C per cycle) 
«FL" ]4taiin 



Fig. 2. Pulse-field gel electrophoresis of HCoV 229E RT-PCR products. Five microliters of 
each PCR reaction were separated by PFGE together with a 5-kbp DNA ladder and a high- 
molecular weight DNA marker (Life Technologies). Also shown are the cycle profiles that 
have been used to produce RT-PCR products ranging from 1 1.5-20.3 kbp in length. 



Table 1 
Oligonucleotide Primers 



Oligonu- 
cleotide 



Sequence (5' to 3') fl 



Position'' 


Orientation' 


Application 


12979-13000 


— 


RT 


21747-21769 


- 


RT 


9071-9091 


+ 


PCR 


20554-20582 


- 


PCR 


293-315 


+ 


PCR 


12830-12850 


- 


PCR 


3860-3877 


+ 


PCR 


21353-21378 


- 


PCR 


1048-1072 


+ 


PCR 






85 ACACACGGTGTATGTCCTCATT 

32 TATAGGCATTGCGCAACCACCGG 

1 27 cgatcgcggccgctggccgaataggccatgGCTGATTACCGTTGCGCTTGT 

1 1 gagaggatccGCAAAACAAACATTTTATTTAGTTGAGAC 

159 cgatcgcggccgctggccgaataggcc ATGGCCTGC AACCGTGTGAC ACT 

89 TCATGGTGTATTTAGTAAGAT 

147 AAACCAGTCTGCTCATCA 

35 tgggacgTCAAAGGACAACTGGTCACATCTCAG 

36 TTGGTCTGTTGGTGATTGGACCGGT 



"The nucleotides corresponding to HCoV 229E sequences are shown in capitals. 

The nucleotides shown in small case were added for cloning purposes. 
*The position refers to the nucleotide sequence of HCoV 229E genomic RNA. 
'Oligonucleotides with mRNA orientation are designated as +. 



Long Distance RT-PCR 65 

coronavirus infection. The hybridization analysis indicates that the material prepared 
using the oligo(dT) 25 magnetic beads (lane 2) is less degraded than the material prepared 
using poly(U)-Sepharose (lane 1). RT-PCR amplifications of more than 5 kbp were only 
possible when poly(A)-containing RNA shown in lane 2 was used as the template for 
reverse transcription. 

5. The oligo(dT) 25 magnetic beads can be regenerated according to the manufacturer's 
instructions (Dynal). However, during the repeated washing steps, you will loose about 
20% of the magnetic beads. 

6. Add the reagents in the order listed in Subheading 3.2. To melt RNA structures before 
the reverse transcription, some protocols recommend to heat the RNA for 10 min at 70°C. 
However, in most cases this is not necessary. 

7. Because the RT is performed at 42°C, cDNAs can be generated during the RT reaction by 
"less stringent" priming events. This is the reason for most of the background PCR prod- 
ucts observed in our system. Therefore, it is absolutely necessary to use a highly specific 
RT -primer and to adjust the optimal primer concentration in the RT-reaction (6). Further- 
more, we recommend the use of different primers for the RT and PCR reactions. 

8. Before adding the Superscript II enzyme, the RNA template and the RT-primer should be 
at 42 C C to minimize unspecific binding. 

9. We recommend incubation for 90 min when cDNA synthesis of more than 10 kb is desired. 
Increasing the incubation temperature above 42°C was not beneficial in our hands. 

10. Some protocols for the amplification of long mRNAs have required digestion of the RNA 
with RNase H after cDNA synthesis (11). This step did not seem to be necessary for 
amplification with HCoV 229E genomic RNA. This may be owing to the fact that in our 
protocols the HCoV 229E RNA was relatively abundant. Experiments with dystrophin 
mRNA required treatment with RNase H prior to amplification (6). 

1 1 . The PCR cycle conditions have to be optimized according to the amount of template, the 
PCR primers and the cycle profile. We recommend that these parameters should be opti- 
mized with RT-PCRs of expected product sizes below 5 kbp before trying to synthesize 
longer RT-PCR products. 

12. This will result in a Mg 2+ concentration of 2 mM. 

13. Optional: a "real" hot start can be performed by adding the enzyme mix after the PCR 
sample has reached 94°C. 

Acknowledgments 

The authors would like to thank A. Rashtchian for helpful discussions and provid- 
ing Superscript II reverse transcriptase and Elongase Enzyme Mix. This work was 
supported by a Grant (SFB 165/B1) from the German Research Council (DFG) to 
SGS. 

References 

1. Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high 
yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216-2220. 

2. Cheng, S., Fockler, C, Barnes, W. M., and Higuchi, R. (1994) Effective amplification of 
long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. USA 91, 
5695-5699. 

3. Cheng, S., Chang, S.Y., Gravitt, P., and Respess, R. (1994) Long PCR. Nature 369, 684-685. 

4. Cheng, S., Higuchi, R., and Stoneking, M. (1994) Complete mitochondrial genome ampli- 
fication. Nature Genet. 7, 350-351. 



66 Thiel, Herold, and Siddell 

5. Cheng, S., Chen, Y., Monforte, J. A., Higuchi, R., and Van Houten, B. (1995) Template 
integrity is essential for PCR amplification of 20- to 30-kb sequences from genomic DN A. 
PCR Meth. Appl. 4, 294-298. 

6. Thiel, V., Rashtchian, A., Herold, J., Schuster, D. M., Guan, N., and Siddell, S. G. (1997) 
Effective amplification of 20-kb DNA by reverse transcription PCR. Analyt. Biochem. 
252, 62-70. 

7. Herold, J., Raabe, T., and Siddell, S. (1993) Molecular analysis of the human coronavirus 
(strain 229E) genome. Arch.Virol. [Suppl] 7, 63-74. 

8. Raabe, T., Schelle-Prinz, B., and Siddell, S. G. (1990) Nucleotide sequence of the gene 
encoding the spike glycoprotein of human coronavirus HCV 229E. ./. Gen. Virol. 71, 
1065-1073. 

9. Siddell, S. (1983) Coronavirus JHM: coding assignments of subgenomic mRNAs. 
/. Gen.Virol. 64, 113-125. 

10. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. D., Smith, J. A., and 
Struhl, K. (1987) Current Protocols in Molecular Biology, (Benson Chanda, V., ed.), 
Wiley, New York. 

11. Nathan, M., Mertz, L. M., and Fox, D. K. (1995) Optimizing Long RT-PCR. Focus 17, 
78-80. 



Increasing PCR Sensitivity for Amplification 
from Paraffin-Embedded Tissues 

Abebe Akalu and Juergen K. V. Reichardt 
1. Introduction 

Many common molecular biology techniques including polymerase chain reaction 
(PCR) (1), Southern blotting (2), comparative genomic hybridization (3), and in situ 
hybridization (4) have been adapted for use with paraffin-embedded tissue (PET). 
PCR-amplified products from PET can be used for, among others, the analysis of loss 
of heterozygosity (1), gene amplification (5), direct sequencing, cloning, and charac- 
terization of genes (6). 

Fixed and embedded material is suboptimal for PCR amplification because of the 
poor quality of extracted genomic DNA. The integrity of the DNA from PET is criti- 
cally dependent on a multitude of factors, including the fixative used, fixation time, 
embedding process, and storage time. Fixation-induced DNA degradations occur as a 
result of extensive cross-linking of proteins to DNA and acid depurinization of the 
DNA, especially for formalin-based fixatives (6,7). As a result, the DNA is often 
nicked, yielding relatively short PCR fragments. In addition, PCR inhibitors (histo- 
logical stains and preservatives) that are coextracted from PET can either cause failure 
of PCR amplifications or greatly reduce the yield of PCR products (8). 

Several approaches and methods have been reported to improve PCR amplification 
from PET. These include dilution of samples (50-100 cells per 1 L of extraction buffer) 
and boiling of extracts (8), phenol/chloroform extraction (9), optimizing the fixation 
and staining process (10), optimization of PCR conditions by prolonging the anneal- 
ing and extension times, reamplification of DNA from certain samples, and stringent 
primer selection (//). Each of these approaches has limitations, and determining the 
optimum conditions for each sample can be laborious and challenging. 

Recently, we have reported an improved method for PCR amplification from PET 
(12). This method uses DNA purified with the QIAquick™ gel extraction kit followed 
by amplification with AmpliTaq Gold®. The combination of these two approaches has 
allowed the routine amplification of PCR fragment up to 959 bp from PET, which 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

67 



68 Akalu and Reichardt 

exceeds the previously expected upper limit of 800 bp (6). The advantages of these 
approaches over the conventional methods are as follows: 

1. The use of QIAquick™ kit for purification is suitable for management of a large number 
of samples by eliminating time-consuming step and hazards of organic extractions. In 
addition, the yield of DNA recovery with the kit is substantially higher than that from 
phenol/chloroform extraction. This variation is possibly attributed to the loss of samples 
during organic extraction and subsequent ethanol precipitation. The alternative approach 
given here is particularly useful when the starting material is small for purification of 
microdissected PET from a single slide. 

2. The gradual activation of AmpliTaq Gold® during thermal cycling allows for high-fidel- 
ity and higher-throughput PCR amplification from PET in which the quality and quantity 
of DNA template can be poor. The reduction of the pre-PCR activation step from 9 to 
5 min and the increase in the number of PCR cycles further enhance the time release PCR 
reaction because polymerase activity builds as specific PCR product accumulates. 

This chapter presents an improved strategy for the preparation of PET DNA for use 
in PCR amplifications including the deparaffinization of PET and extraction of DNA. 
Because the QIAquick™ kit is primarily designed for isolation of DNA from agarose 
gels, the adaptation of this kit for DNA purification from PET is also outlined. In 
addition, a simplified microdissection protocol is presented, as there is an increasing 
interest in using microdissected PET for the analyses of molecular events leading to 
malignant transformation step. Finally, the essential aspects of PCR parameters using 
AmpliTaq Gold® for amplification from PET are described. 

2. Materials 

2.1. Deparaffinization of PET Block 

1. PET blocks (see Note 1). 

2. Absolute ethanol. 

3. Xylene (see Note 2). 

4. Scalpel or razor blade. 

2.2. Microdissection of PET Section from Slides 

1. Stained or unstained slides containing tissue sections from PET (see Note 1). 

2. 30-gauge needle. 

3. Cover slip. 

4. 50% glycerol (diluted with sterile water). 

5. Microscope. 

2.3. Digestion of PET with Proteinase K 

1. Digestion buffer: 10 raM Tris-HCl (pH 8.0), 1 mM ethylenediaminetetraacetic acid 
(EDTA), and 1% Tween-20. 

2. Proteinase K (20 mg/mL stock solution). 

3. Water bath. 

2.4. Purification of DNA from PET 

1. TE buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. 

2. 100% Isopropanol. 

3. QIAquick™ Gel Extraction Kit (Qiagen, Chats worth, CA). 



Increasing PCR Sensitivity 69 

2.5. PCR Amplification from PET 

1 . 100 mM dNTP stock solution (Gibco-BRL® Rockville, MD). Prepare a mixture of 10 mM 
of each dNTP (dGTP, dATP, dCTP, dTTP) in sterile water. 

2. AmpliTaq Gold® Kit (Perkin-Elmer, NJ) containing 10X buffer II, 25 mM MgCl 2 , and 
AmpliTaq Gold® (5 U/uL). 

3. Purified DNA sample or crude extract from PET. 

3. Methods 

3.1. Microdissection of PET 

Selective procurement of histopathologically defined cell populations from stained 
or unstained tumor sections on glass slides can be microdissected to study the molecu- 
lar genetic events that drive the multistep transformation in tumors. Several kinds of 
microdissection techniques have been described with the development of multiplex 
molecular analysis using PCR technology (4,13,14). The following simplified proto- 
col is routinely used in our laboratory for microdissection from deparaffinized and 
stained slides (see Notes 3 and 4; see Fig. 1): 

1. Place a drop of 50% glycerol on the area to be selectively microdissected from the tissue 
section on a slide. 

2. Place a glass cover slip over the drop of glycerol under a light pressure in order to moisten 
and ease detachment of the cells from the slide. After 5-10 min, carefully lift cover slip 
off with a scalpel. 

3. Place the slide under a microscope and identify the selected area. Cells can be easily 
identified using a lOOx magnification or wide-field microscopy such as an inverted 
microscope for cell culture. 

4. Scratch selected cells, which were identified by microscopic visualization (see Notes 
5-7), with a disposable sterile 30-gauge needle. 

5. Transfer the tissue fragments adhering to the tip of the needle into a 1.5-mL 
microcentrifuge tube containing digestion buffer (see Note 8). 

6. Repeat steps 4 and 5 until the area is entirely microdissected and detached tissues are 
removed. Proceed with Subheading 3.3. 

3.2. Deparaffinization of PET Blocks (see Note 9) 

1. If a microtome is available cut paraffin blocks into single or multiple 10-u.m sections (see 
Note 10). If no microtome is available, using a razor blade or disposable scalpel, cut a 
small section of PET blocks on a clean surface and then transfer the sample into a 1.5-mL 
microcentrifuge tube using a sterile toothpick or forceps. Sections may be minced into 
small pieces with scalpel for ease of transferring into a microcentrifuge tube. 

2. Add 1 mL xylene and vortex until the paraffin wax sections dissolve. 

3. Centrifuge at 10,000g for 3 min at room temperature to pellet the tissues. 

4. Carefully remove wax/xylene supernatant into a waste bottle by pipeting. 

5. Resuspend the tissue pellet in 1 mL of 100% ethanol by gently vortexing and then centri- 
fuge at 10,000g for 3 min at room temperature. Remove the supernatant. 

6. Repeat step 5. Air-dry the tissue pellet at room temperature until the ethanol evaporates 
completely. 

7. Resuspend the pellet with digestion buffer and proceed with Subheading 3.3. 



70 



Akalu and Reichardt 



1 . Sample preparation. 



X 



PET section slides. 
I 



r 

Deparaffinized tissue. Embedded tissue. 

♦ t 

Microdissect selected areas. Microdissect selected areas.,, 

t 

Deparaffmize tissue. 

t 



1 

PET blocks. 

I 

Cut block into small pieces. 



2. DNA extraction with proteinase K digestion. 



3. DNA purification with QIAquick kit. 



4. PCR amplification with AmpliTaq Gold. 



Fig. 1. General overview of processing PET for PCR amplification. After DNA extraction 
with proteinase K digestion, purification of DNA from PET may not be necessary if the target 
sequence is short (usually less than 300 bp) and optimal primers, PCR conditions, and poly- 
merase are used. In this case, PCR amplification can be tried directly from crude tissue extracts. 



3.3. Digestion of Tissue with Proteinase K 

1. Add digestion buffer into 1.5-mL microcentrifuge tube containing deparaffinized tissue 
pellet from PET blocks or microdissected tissues. The volume of the buffer usually ranges 
50-200 (xL depending on the amount of tissues (see Notes 7 and 10). Boil the tube for 
3 min and suspend tissues by gently vortexing. Add 100 (ig/mL proteinase K to the diges- 
tion buffer and incubate the tube at 52°C for 12-14 h. 

2. Inactivate the proteinase K by boiling the tube for 10 min. 

3. The extracted DNA can be stored at -20°C until it is used. 

3.4. Purification of DNA 

Purification is suggested to remove coextracted stains, residual fixation chemicals, 
and proteins. It is also possible to remove most of the degraded DNAs that compete 
with intact target sequences for dNTPs and primers during purification. The 
QIAquick™ Gel Extraction Kit is a silica-based technology used for isolation of DNA 
fragments (70 bp to 10 kb) from agarose gels as well as for DNA cleanup from enzy- 
matic reactions (see Note 11). We adapted this kit also for the purification of DNA 
extracts from PET as follows (12): 



Increasing PCR Sensitivity 71 

1 . Add 4-5 volumes of binding buffer QG to DNA extract from PET. 

2. Add one volume of isopropanol to DNA extract and mix gently by pipeting. 

3. Place the QIAquick™ spin column in a 2-mL tube. Load the sample from step 2 into the 
spin column. Centrifuge at 5000g for 1 min. Discard the flowthrough. 

4. Place the spin column back into the same 2-mL tube. Add 0.75 mL of wash buffer PE and 
incubate the column for 5 min at room temperature. Centrifuge at 5000g for 1 min. Dis- 
card the flowthrough. 

5. Place the spin column into the same tube. Centrifuge at lOOOg for 1 min. 

6. Transfer the spin column into a new 1.5-mL recovery tube. Add 30-100 uL of TE buffer 
directly to the center of the spin, incubate for 3 min at room temperature, centrifuge at 
10,000g to elute the DNA, and then store at -20°C until it is used. 

3.5. PCR Amplification from PET 

Set up the PCR reaction (50 [xL/reaction) by adding the AmpliTaq Gold® reagents 
into 0.5-mL microcentrifuge tube as follows: 

1. 5 uL of 10X PCR buffer II (100 mM Tris-HCl, pH 8.3, 500 mM KC1). 

2. 4 uL of 25 mM MgCL (final concentration 2 mM). 

3. 4 uL of 10 mM dNTPs (final concentration 200 \iM of each dNTP). 

4. 0.1-0.2 \iM of each forward and reverse primer (see Note 12). 

5. 5-20 ng of genomic DNA (see Note 13). 

6. 2.5 U AmpliTaq Gold®/reaction. 

7. Bring to a total volume of 50 uL with sterile water. 

8. Overlay the reaction mixture with 1 drop of sterile mineral oil. 

9. Amplify the PCR reaction in a programmable thermal cycler. The following thermal 
cycling profile in RoboCycler® Gradient 40 (Stratagene, La Jolla, CA) is optimized for 
the amplification of fragments shown in Fig. 2: 

a. Step 1: 1 cycle of initial denaturation at 95°C for 5 min (see Note 14). 

b. Step 2: 60 cycles of denaturation at 95°C for 2 min, annealing at 60°C for 80 s, and 
elongation at 72°C for 80 s (see Note 15). 

c. Step 3: 1 cycle of final extension at 72°C for 5 min. 

4. Notes 

1. For PCR amplifications, prepared specimen of PET on glass slides or as paraffin blocks 
are usually obtained from a pathology laboratory. 

2. Xylene is a toxic organic solvent. Octane or commercially available solvents such as 
Hemo-De® Clearing Agent (Fisher Scientific) or AmeriClear® (Baxter Scientific) can be 
substituted. 

3. Unstained sections on slide can also be used as long as the areas of interest are identified. 
The areas of tumors to be microdissected are selected and circled with a felt-tip pen on the 
inverted side of the slide usually by a pathologist. 

4. A good morphological distinction between benign and malignant cells usually requires 
histologic staining of tissues for microscopic visualization. Usually sections are stained 
after deparaffinization. Sometimes, tissue sections on slides are stained without prior 
deparaffinization since paraffin holds the tissue fragments together during the microdis- 
section process (10). In this case, sections are deparaffinized after microdissection. To 
deparaffinize microdissected tissues follow steps 2-7 in Subheading 3.2., by using less 
xylene (300 uL) and ethanol (300 uL). 



72 



Akalu and Reichardt 



S59 dp ► 




Fig. 2. An example of amplification from PET following the PCR parameters in Subhead- 
ing 3.5. The tumor tissues were microdissected from deparaffinized and stained slide, digested 
with proteinase K, and purified with QIAquick™ kit. About 20 ng of DNA template was used 
for a PCR amplification with AmpliTaq Gold®. Lanes 1, 2, and 3 show amplification of 959 bp, 
521 bp, and 309 bp fragments, respectively. M, DNA ladder. This figure is modified from 
ref. 12 with permission from Elsevier Science. 



5. 



6. 



10. 



11. 



Given the heterogeneity of biopsies, and the infiltrative nature of many tumors, an impor- 
tant consideration in microdissection is the minimization or possibly eliminating the con- 
tamination of normal cells with neoplastic cells or vice versa. 

For the purpose of analyzing loss of heterozygosity from the same slide, normal tissues adja- 
cent to the tumor cells should be microdissected and placed in a separate tube for analyses. 
The size of tumor area is variable usually ranging 5-20 mm 2 . If the size of the tumor is 
small, the same specific region from duplicate tissue slides can be microdissected, and 
scrapes can be pooled in one microcentrifuge tube to give sufficient DNA. Thus, the 
volume of the digestion buffer can range from 50-200 u,L depending on the size of the 
tumor and the number of slides to be microdissected. 

Microdissected samples from deparaffinized tissue slides are directly scraped off into 
1.5-mL microcentrifuge tube containing digestion buffer. From undeparaffinized slides, 
tissues are scraped off into tube containing xylene, and then deparaffinized {see Note 4). 
Deparaffinization of PET is a widely used procedure to promote digestion of tissues with 
proteinase K and PCR amplification. However, it is reported that deparaffinization may 
not be necessary for PCR analysis of RNA from PET (15). 

For a number of analyses, tissue sections (5-10 u,m) are prepared from PET blocks and 
mounted on slides. Sectioning of paraffin blocks using a microtome requires training and 
experience. The amount of sections required for extraction depends on the availability 
and the intended use of the tissue. If more tissue is required, a section of blocks 50-100 
mg (or more) can be cut and processed. Do not exhaust all the blocks. 
Recently, a DNeasy™ Tissue Kit for purification of DNA from PET has been introduced. 
In our laboratory, no differences in the quality and quantity of purified DNA for PCR 



Increasing PCR Sensitivity 73 

amplification were observed between DNeasy™ Tissue Kit and QIAquick™ Gel Extrac- 
tion Kit. Because the gel extraction kit is routinely used for isolation of DNA from agar- 
ose gels for cloning, the use of the kit for DNA purification from PET is an added benefit. 

12. As always, primer design is one of the most important aspects of PCR. Because DNA 
template from PET is a complex mixture of degraded DNA fragments, careful attention is 
required to designing primers for PCR amplification from PET. Not all primers that 
amplify from fresh tissue can amplify fragments from PET. Although common guidelines 
are available, software programs such as Primer3 (from S. Rozen, H. and J. Skaletsky, 
http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) can be used for optimum 
primer selection. In the event of failure to amplify fragments, it usually helps to try differ- 
ent sets of primer pairs. It is also helpful to screen extracts from PET for the integrity and 
quantity of amplifiable DNA by running a control amplification reaction for a single- 
copy housekeeping gene such as |3-actin primer (16) or another suitable target. 

13. The critical factor for successful PCR amplification from PET is the integrity of the 
target sequence. The amount of usable genomic DNA extracted from PET depends on 
multiple factors, including the amount of tissue, storage time, the chemical composi- 
tion of the fixative, fixation time, and the embedding process. In addition, yields of 
genomic DNA will vary from tissue to tissue from which the DNA is extracted. The 
amount of DNA is greater in tissues containing concentrated nucleated cells than tis- 
sues with fewer nucleated cells. DNA extraction from PET in most cases yields approx 
10 ng of DNA from 1000 cells. Determination of the concentration of DNA extracted 
from PET may not be necessary, if the amount of starting material is too small. This 
may also help in saving irreplaceable DNA sample. However, if the DNA is extracted 
from large blocks of PET, the concentration can be determined following a standard pro- 
cedure. From our experience, 1-2 uE of purified DNA from microdissected tissue with an 
area of 5-20 mm 2 gives a reasonable yield of PCR products. In some cases, if the DNA is 
either concentrated or crude cell extract is used for amplification, appropriate dilution of 
the template should be made. 

14. AmpliTaq Gold® is reversibly chemically inactivated. It gains about 40% of its activity 
with a pre-PCR heat step of 95°C for 9-12 min and its reactivation continues with subse- 
quent thermal cycles in a time-release manner. The manufacturer's recommended time 
(at 95 °C for 9-12 min) should be reduced to 2-5 min in order to increase the time-release 
effect of AmpliTaq Gold®. To compensate for the shortening of the precycling heat step, 
increased thermal cycles up to 50 or more are required so that enough PCR products is 
generated for restriction analysis, sequencing, and cloning. 

15. The number of thermal cycles is determined by the amount of input DNA and intended 
use of the PCR product for analysis. For instance, when [y- 32 P] ATP radiolabeled primers 
are used for screening mutations and genotyping by single-strand conformation polymor- 
phism analysis, the number of thermal cycles should not exceed 30 and the preincubation 
of 95 °C for 9 min can be used. Because the method is so sensitive, radiolabeled PCR 
products from over 30 cycles usually show nonspecific amplifications and high back- 
ground on autoradiography. This will largely compromise the results and lead to incor- 
rect genotyping. 

Acknowledgments 

This work was supported by grants from the DoD for the USC Prostate Center 
(PC 992018; project A) and the TJ Martell Foundation to JKVR. 



74 Akalu and Reichardt 

References 

1. Akalu, A., Elmajian D. A., Highshaw, R. A., Nichols, P. W., and Reichardt, J. K.V. (1999) 
Somatic mutations at the SRD5A2 locus encoding prostatic steroid 5ct-reductase during 
prostatic cancer progression. ,/. Urol. 161, 1355-1358. 

2. Bramwell, N. H. and Burns, B. F. (1988) The effects of fixative type and fixation time on 
the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue. 
Exp. Hematol. 116, 730-732. 

3. Speicher, M. R., Jauch, A., Walt, H., du Manoir, S., Schrock, E., Holtgreve-Grez, H., 
Schoell, C, Lengauer, C, Cremer, T., and Reid, T. (1993) Molecular cytogenic analysis 
of formalin-fixed, paraffin embedded solid tumors by comparative genomic hybridiza- 
tion. Hum, Mol. Genet. 2, 1907-1914. 

4. Nuovo, G. J. and Silverstein, S. J. (1988) Methods in laboratory investigation: comparison 
of formalin, buffered formalin, and Bouin's fixation on the detection of human 
papillomavirus deoxyribonucleic acid from genital lesions. Lab. Invest. 59, 720-724. 

5. Ribot, E. M., Quinn, F. D., Bai, X., and Murtagh, J. J., Jr. (1998) Comparative PCR: An 
improved method to detect gene amplification. BioTechniques 24, 22-26. 

6. Crisan, D. and Mattson, J. C. (1993) retrospective DNA analysis using fixed tissue speci- 
mens. DNA Cell Biol. 12, 455-464. 

7. Jacson, V. (1978) Studies on histone organization in the nucleosome using formaldehyde 
as a reversible cross-linking agent. Cell 15, 945-954. 

8. Zhuang, Z., Bertheau, P., Emmert-Buck, M. R., Liotta, L. A., Gnarra, J., Linehan, W. M., 
and Lubensky, I. A. (1995) A microdissection technique for archival DNA analysis of 
specific cell populations in lesions < 1 mm in size. Am. J . Pathol. 146, 620-625. 

9. Greer, C. E., Peterson, S. L., Kiviat, N. B., and Manos, M. M. (1991) PCR amplification 
from paraffin-embedded tissues. Effects of fixative and fixation time. Am. J . Clin. Pathol. 
95, 117-124. 

10. Burton, M. P., Schneider, B. G., Brown, R., Escamilla-Ponce, N., and Gulley, M. L. (1997) 
Comparison of histologic stains for use in PCR analysis of microdissected, paraffin- 
embedded tissues. BioTechniques 24, 86-92. 

11. Wright, D. K. and Manos, M. M. (1990) Sample preparation from paraffin-embedded tis- 
sues, in PCR Protocols, A Guide to Methods and Applications (Innis, M. A., Gelfand, D. 
H., and Sninsky, J. J., eds.) Academic, San Diego, CA, pp. 32-38. 

12. Akalu, A. and Reichardt, J. K. V. (1999) A reliable PCR amplification method for 
microdissected cells obtained from paraffin-embedded tissue. Genet. Analyt. 15, 229-233. 

13. Walch, A., Komminoth, P., Hutzler, P., Aubele, M., Hofler, H., and Werner, M. (2000) 
Microdissection of tissue section: application to the molecular genetic characterization of 
premalignant lesions. Pathology 68, 9-17. 

14. Baisse, B., Bian, Y.-S., and Benhattar, J. (2000) Microdissection by exclusion and DNA 
extraction for multiple PCR analyses from archival tissue sections. BioTechniques 28, 
856-862. 

15. Shimizu, R. and Burns, J. C. (1995) Extraction of nucleic acids: sample preparation from 
paraffin-embedded tissues, in PCR Strategies (Innis, M. A., Gelfand, D. H., and Sninsky, 
J. J., White T. J., eds.), Academic, San Diego, CA, pp. 153-136. 

16. Benz-Ezra, J., Johnson, A., Rossi, J., Cook, N., and Wu, A. (1991) Effect of fixation on 
the amplification of nucleic acids from paraffin-embedded material by the polymerase 
chain reaction. J. Histochem. Cytochem. 39, 351-354. 



8 

GC-Rich Template Amplification by Inverse PCR 

DNA Polymerase and Solvent Effects 

Alain Moreau, Da Shen Wang, Steve Forget, Colette Duez, 
and Jean Dusart 

1. Introduction 

The amplification of GC-rich templates by any PCR method is usually a difficult 
task and despite the development of modified methods and conditions, this type of 
amplification still remains a specific case approach. Problems usually observed with 
GC-rich DNA are constraint of template amplification by stable secondary structures 
that stall or reduce the DNA polymerase progress, and the presence of secondary 
annealing sites giving rise to nonspecific amplified bands. This latter point is not 
exclusive to GC-rich templates but is frequently encountered in other types of tem- 
plates. In order to design a more general method for GC-rich templates, different DNA 
polymerases were compared in combination with different organic solvents with the 
purpose of abolishing stable secondary structures (1). Our attention focused on the 
inverse polymerase chain reaction (iPCR) used to perform site-directed mutagenesis 
(1,2). This very attractive method requires a single pair of primers and involves the 
amplification of the whole recombinant plasmid, a difficult step with high GC-content 
DNA. Inverse PCR also proves useful in cloning missing parts of genes by using a 
self-ligated genomic DNA fragment as template. 

A recent survey of the literature showed the absence of comparative studies regard- 
ing the use of different DNA polymerases in the amplification of GC-rich DNA with 
or without the addition of organic solvents, such as dimethyl-sulfoxide (DMSO) (3,4), 
formamide (3,5,6), and tetramethylammonium chloride (TEMAC) (7), Furthermore, 
little is known of the exact role of these chemicals in PCR. It was suggested that these 
compounds primarily affect the annealing kinetics as well as the efficiency of the 
DNA polymerase used. In order to identify critical parameters involved in iPCR with 
GC-rich templates, we analyzed the influence of DNA polymerases in combination 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

75 



76 Moreau et al. 

with the aforementioned solvents (1). The results obtained allowed us to improve iPCR 
for difficult template amplifications by either iPCR or standard PCR. Our iPCR method 
can be divided into two steps: amplification of the whole recombinant plasmid and 
iPCR product purification and ligation. This method was used to perform site-directed 
mutagenesis by amplification of a 4.8-kb plasmid derived from pUC 1 8 and containing 
a 1980-bp insert, the gene encoding the extracellular DD-carboxypeptidase from 
Actinomadura R39, a 74% GC-content DNA (14). Different DNA polymerases were 
tested according to the manufacturer's specifications. However, correct amplification 
was not detected with any DNA polymerase tested (7). The efficiency of amplification 
by addition of DMSO, formamide, or TEMAC in the reaction mixture was evaluated 
according to the conditions described above. Analysis of PCR products in the pres- 
ence of these organic solvents revealed that only Vent™ DNA polymerase (New 
England Biolabs, Berverly, MA) amplified the 4.8-kb plasmid (1). We focused our 
attention on the conditions to amplify GC-rich DNA templates with Vent DNA poly- 
merase. In addition, the Mg 2+ concentration was increased to obtain a 10-mM final 
concentration. This allowed us to amplify large DNA fragments in the PCR assay. 

2. Materials 

1. pBlueScript™ vector (Stratagene, La Jolla, CA) or any other pUC derivative plasmid 
used to clone the gene to be mutated (see Note 1). 

2. 25-50 ng of DNA template (see Note 2). 

3. 2 \\.M of each oligonucleotide, primers A and B corresponding respectively to the sense 
(coding) and antisense (noncoding) message (see Note 3). 

4. 10X DNA polymerase buffer (provided by the manufacturer of the Vent DNA poly- 
merase) (see Note 4) and Vent DNA polymerase (see Note 5). 

5. dNTPs: Mix 10 mM each (Pharmacia LKB, Piscataway, NJ). 

6. 100mMMgSO 4 . 

7. Fresh, deionized formamide (Sigma, St. Louis, MO). 

8. Sterile water. 

9. Light mineral oil (Sigma). 

10. Reagents for agarose gel electrophoresis (Life Technologies [Gibco-BRL], Gaithers- 
burg, MD). 

1 1. Sephadex G-50 fine (Pharmacia LKB), siliconized wool and 1-mL syringe. 

12. T4 polynucleotide kinase (PNK) (New England Biolabs). 

13. Reagents for ligation: 10X ligation buffer (Boehringer Mannheim, Indianapolis, IN); T4 
DNA ligase (Boehringer Mannheim, Germany). 

14. 400-uL and 1.5-mL sterile Eppendorf tube. 

15. Thermocycler. 

3. Methods 

3.1. Inverse PCR 

1. Perform iPCR by adding in a 400-uL sterile Eppendorf tube the following reagents (see 
Note 6): 5 uL DNA template (25-50 ng); 10 uL 10X Vent DNA polymerase buffer; 8 uL 
MgS0 4 (100 mM); 2 uL dNTPs (10 mM each); 4 uL primer A (50 pmol/uL); 4 uL primer 
B (50 pmol/uL); 10 uL formamide; 56 uL sterile H 2 0; and 1 uL Vent DNA polymerase 
(2 U/uL); to a total volume of 100 uL. 



Template Amplification by iPCR 77 

2. Overlay the sample with 30 \xL of light mineral oil. 

3. Submit the samples to a standard three-step cycling protocol according to the following 
parameters (see Note 7): 95°C for 1 min (initial denaturation) (1 cycle); 94°C for 30 s 
(denaturation); XX C C, 1 min (annealing); 72°C for Y min (extension) 30 cycles; 72°C for 
10 min (final extension) (1 cycle). 

3.2. iPCR Product Purification and Ligation 

1 . Separate the iPCR reaction from the light mineral oil by simply pipeting only the reaction 
volume from the bottom of the tube into a new 1 .5-mL sterile Eppendorf tube. 

2. Take 10-20 (xL aliquot of the iPCR reaction to visualize the product by agarose gel elec- 
trophoresis (0.7% agarose, see Note 8). 

3. Purify the iPCR product by passing the remaining iPCR reaction through a Sephadex G- 
50 spun column of 1-mL and elute with 100 u.L of TE buffer or H 2 0. 

4. Perform the phosphorylation and ligation reaction by adding the following reagents in a 
1.5-mL sterile Eppendorf tube: 10 ^L aliquot of purified iPCR reaction; 2 [xL 10X liga- 
tion buffer; 0.5 [xL T4 polynucleotide kinase (10 U/[xL); and 7.5 [xL H 2 sterile; to a total 
volume of 20 \iL. 

5. Incubate the mixture 15 min at 37°C, then again add 0.5 jxL of T4 polynucleotide kinase 
and incubate for another 15 min at 37°C. 

6. Add 1 [xL of T4 DNA ligase (1 U/[xL) to the reaction mixture and incubate overnight at 
4°C, followed by 16°C for 3 h. 

7. Transform competent Escherichia coli (E. coli) cells with 5-(xL aliquot of the ligation 
mixture. 

4. Notes 

1. The targeted DNA is initially cloned in a vector, in general, a pUC-derived plasmid. 
Because the difficulty of amplification increases with plasmid length, it is better to avoid 
unnecessarily large plasmid and clone only a part of the gene flanking the targeted DNA. 
Once mutated by iPCR, this part will then be used to reconstruct the whole gene. The 
latter step reduces the subsequent sequencing necessary to check the integrity of the 
mutated DNA fragment. 

2. The template concentration used to perform iPCR is about 25-50 ng. Higher concentra- 
tions will increase the background because of the wild-type plasmid that easily trans- 
forms E. coli. Lower template concentration reduces the final amount of amplified 
material, thus requiring more PCR cycles. These additional cycles contribute to the intro- 
duction of more errors by DNA polymerases. Under our conditions, 30 cycles are suffi- 
cient to amplify GC-rich templates. 

3. The primer design is a crucial step. In iPCR, the primers to be used are oriented in 
inverted tail-to-tail direction, i.e., one primer corresponding to the coding sense (5-3') 
whereas the other is antisense (3'-5'). Usually, one primer harbors the mutation, which 
can be a substitution, deletion, or insertion of one or more nucleotides. The selection of 
this pair of primers is sometimes difficult, but can be simplified by using one of several 
new computer programs for oligonucleotide selection (12,13). However, this initial step 
is frequently overlooked, resulting in great difficulties in template amplification (not just 
GC-rich ones). There are three basic rules to follow to avoid primer design problems: 

a. Elimination of duplex formation at the 3' ends with either one or both primers, plus 
elimination of hairpin structure formation within primers. 

b. Design of primers with T m (°C) close to each other, i.e., less than a 10 C C difference. 
The addition of any organic solvent to the PCR reaction will decrease the T m for a 



78 Moreau et al. 

specific primer; and its partner primer bearing the mutation will also show reduced 
T m depending on the introduced mutation, 
c. Location of the chosen mutation in the middle of the primer in order to maintain the 
internal stability of the oligonucleotide. 

4. The buffer supplied by the manufacturer is IX: 20 mM Tris-HCl, pH 8.8 (at 25°C), 10 
mM KC1, 10 mM (NH 4 ) 2 S0 4 , 2 mM MgS0 4 , and 0.1% Triton X-100. 

5. The choice of DNA polymerase is a key point for the amplification of the whole recombi- 
nant plasmid. Among several DNA polymerases tested, our choices were Vent DNA poly- 
merase (8-10) and Pfu DNA polymerase (Stratagene) (11). Indeed, these two enzymes 
produce almost exclusively blunt ends, whereas Taq DNA polymerase requires additional 
manipulations to obtain blunt ends. Furthermore, Vent and Pfu DNA polymerase are more 
accurate during iPCR than Taq DNA polymerase. We recommend testing the first iPCR 
reactions without the addition of organic solvents. It is difficult to choose the solvent and 
its optimal concentration without empirical assay. Furthermore, it has been noted that 
most DNA polymerases are sensitive to organic solvents, especially formamide. How- 
ever, the use of 10% formamide is suggested in combination with Vent DNA polymerase 
(1). This DNA polymerase proved to be the most robust enzyme tested in the presence of 
formamide, because other DNA polymerases {Taq and Pfu) could not amplify DNA under 
similar conditions (Moreau, A., unpublished observations). Another possibility is the use 
of Pfu DNA polymerase without any solvent addition. In some cases, Pfu DNA poly- 
merase gives rise to good amplification with GC-rich templates but does not tolerate 
formamide concentrations greater than 2.5%. 

6. We observed that the Mg 2+ concentration is very important in obtaining proper amplifica- 
tion with the Vent DNA polymerase, especially with large plasmids (5 kb). In the absence 
of amplification products, we recommend the modification of Mg 2+ concentration by 
supplemental addition of increasing amounts of Mg 2+ in the iPCR reaction. The optimum 
Mg 2+ concentration usually occurs in a narrow range with the Vent DNA polymerase. 
Among a choice of several organic solvents, 10% formamide was the most useful addi- 
tion to correctly amplify the 4.8 kb plasmid (see Fig. 1). Higher formamide concentra- 
tions appeared detrimental for the iPCR. It was also observed that addition of <8% DMSO 
failed to amplify the plasmid but higher concentrations did not significantly improve the 
iPCR, even with addition of T4 gene 32 protein, a single-stranded binding protein used to 
overcome secondary structures (15,16). The addition of TEMAC at concentrations from 
10~ 2 -10~ 5 M was not effective (see Fig. 1). The iPCR assay was repeated with different 
pairs of primers, in different locations and each time, the 4.8-kb plasmid was successfully 
amplified. 

7. We choose to perform iPCR with the Actinomadura R39 DD-carboxypeptidase (74% 
GC-rich) with an annealing temperature of 55°C/1 min, and 5 min extension time at 72°C. 
This iPCR has been performed with the following two primers: 5'-GCCCTCGGCG 
GCGGTACCGCAT-3' (T m =70 o C), which anneals perfectly with the target sequence and 
5'-GTGGTCGAGGCCCACACCGGGACGATG-3' (T m =61.6°C) introducing two non- 
contiguous mismatches in the sequence (because these substitutions are not contigu- 
ous, we have considered them as three mismatches). The annealing temperature has also 
been reduced, based on the guideline of T m decrease of both primers by about 0.6°C/% of 
formamide added to the iPCR reaction. We used the extension time of 1 min/kb of plas- 
mid for the polymerization at 72°C, and a final 10-min polymerization at 72°C. The guide- 
line of 1 min/kb for the extension at 72°C is suitable when the plasmid size is 5 kb or less, 



Template Amplification by iPCR 



79 




Fig. 1. Solvent effects on iPCR. A 4.8-kb plasmid containing a GC-rich (74%) 1980-bp insert 
was amplified by iPCR using Vent™ DNA polymerase. Samples covered with mineral oil (30 fxL) 
were submitted to 30 amplification cycles on a Biometra Trio-Thermoblock™ :1 min denatur- 
ation at 95°C, 1 min annealing at 55°C, and 5 min extension at 72°C, followed by a final 
10-min extension at 72°C. A 10-u.L aliquot of each sample was loaded onto a 0.7% agarose gel. 
Lanes 1 and 17, 1-kb DNA ladder size standard; lane 2, control iPCR under standard conditions 
without organic solvent; lanes 3-6, iPCR in presence of 8, 10, 15, and 20% DMSO respectively; 
lanes 7-10, iPCR in presence of 5, 10, 15, and 20% formamide respectively; lanes 11-14, iPCR 
in presence of 10" 2 , 10~ 3 , 10 -4 , and 10~ 5 M TEMAC, respectively; lane 15, iPCR in presence of 
8% DMSO with 3 jig of T4 gene 32 protein; and lane 16, iPCR under standard conditions with 
3 \ig of T4 gene 32. 



whereas with larger plasmid size, the extension time should be increased on an empirical 
basis. The initial denaturation is an important step, but prolonging it may be detrimental 
for longer templates. 

An aliquot of the iPCR reaction is visualized by agarose gel electrophoresis. The pres- 
ence of minor bands of smaller size than the wild-type plasmid is not a real problem. 
Most of them will generate partial plasmids missing key regions of replication or antibi- 
otic resistance and will be easily eliminated after transformation. However, several meth- 
ods are available to purify PCR products. We use a Sephadex G-50 spun-column to 
remove unincorporated dNTPs. A purified aliquot is then phosphorylated and ligated. It 
is not unusual to find frameshifts+1 or -1 at the junction of each primer after isolation of 
plasmid DNA from transformants obtained through iPCR product ligation. However, we 
observed that most of the transformants are the result of correct ligation. 



80 Moreau et al. 

References 

1. Moreau, A., Duez, C, and Dusart, J. (1994) Improvement of GC-rich template amplifica- 
tion by inverse PCR. BioTechniques 17, 233-234. 

2. Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse 
polymerase chain reaction. Genetics 120, 621-623. 

3. Bookstein, R., Lai, C. C., To, H., and Lee, W. H. (1990) PCR-based detection of a poly- 
morphic BamHI site in intron 1 of the human retinoblastoma (RB) gene. Nucl. Acids Res. 
18, 1666. 

4. Winship, P. R. (1989) An improved method for directly sequencing PCR amplified mate- 
rial using dimethyl sulphoxide. Nucl. Acids Res. 17, 1266. 

5. Sarkar, G., Kapelner, S., and Sommer, S. S. (1990) Formamide can dramatically improve 
the specificity of PCR. Nucl. Acids Res. 18, 7465. 

6. Schuchard, M., Sarkar, G., Ruesink, T., and Spelsberg, T. C. (1993) Two-step "hot" PCR 
amplification of GC-rich avian c-myc sequences. BioTechniques 14, 390-394. 

7. Hung, T., Mak, K., and Fong, K. A. (1990) Specificity enhancer for polymerase chain 
reaction. Nucl. Acids Res. 18, 4953. 

8. Eckert, K. A. and Kunkel, T. A. (1991) DNA polymerase fidelity and the polymerase 
chain reaction. PCR Meth. Applicat. 1, 17-24. 

9. Kong, H., Kucera, R. B., and Jack, W. E. (1993) Characterization of a DNA polymerase 
from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady 
state kinetics, thermal stability, processivity, strand displacement, and exonuclease activi- 
ties. ./. Biol. Chem. 268, 1965-1975. 

10. Manila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991) Fidelity of DNA synthesis 
by the Thermococcus litoralis DNA polymerase — an extremely heat stable enzyme with 
proofreading activity. Nucl. Acids Res. 19, 4967-4973. 

11. Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short , J. M., Sorge, J. A., and Mathur, 
E. J. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated 
from Pyrococcus furiosus. Gene 108, 1-6. 

12. Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A. (1986) Predicting DNA duplex 
stability from the base sequence. Proc. Natl Acad. Sci. USA 83, 3746-3750. 

13. Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T., and 
Turner, D. H. (1986) Improved free-energy parameters for predictions of RNA duplex 
stability. Proc. Natl. Acad. Sci. USA 83, 9373-9377. 

14. Granier, B., Duez, C, Lepage, S., Englebert, S., Dusart, J., Dideberg, O., et al (1992) 
Primary and predicted secondary structures of the Actinomadura R39 extracellular DD- 
peptidase, a penicillin-binding protein (PBP) related to the Escherichia coli PBP4. 
Biochem.J. 282, 781-788. 

15. Alberts, B. and Sternglanz, R. (1977) Recent excitement in the DNA replication problem. 
Nature 269, 655-661. 

16. Bittner, M., Burke, R. L., and Alberts, B. M. (1979) Purification of the T4 gene 32 protein 
free from detectable deoxyribonuclease activities. J. Biol. Chem. 254, 9565-9572. 



PCR Procedure for the Isolation 
of Trinucleotide Repeats 

Teruaki Tozaki 
1. Introduction 

Microsatellites, also referred to as short tandem repeats (STR) or simple sequence 
repeats (SSR), are highly polymorphic and abundant sequences dispersed throughout 
most eukaryotic nuclear genomes (1-3). In recent years, microsatellites have been 
used for linkage map construction, population genetics, molecular evolution studies, 
forensic sciences, and as parentage testing markers (4,5). 

Trinucleotide repeats, such as (CAG) n and (CGG) n repeats, have drawn increasing 
attention in various aspects of both human gene mapping and clinical genetics. Genes 
with trinucleotide repeats, such as transcription-regulatory proteins and homeobox 
genes, are frequently found in mammalian genomes (6). Some of these genes are in- 
volved in neuropsychiatric disorders in human, such as Huntington's disease (7) and 
spinocerebellar ataxia type 1 (8) . At present, the function of the trinucleotide repeats is 
not clear. 

Polymorphism of trinucleotide repeats can be resolved more easily in alleles differ- 
ing by one repeat than those of dinucleotide repeats. Thus, tri- and tetranucleotide 
repeats are useful for human forensic sciences. The abundance and polymorphic infor- 
mation of trinucleotide repeats in the human genome have been characterized by 
Gastier et al. (9). In the human genome, (CAG) n repeats occur, on average, once every 
4400 kb, making them less abundant than dinucleotide repeats. Thus, it is considered 
that the construction of an enrichment library is useful for the isolation of trinucleotide 
repeats. 

A novel, rapid, and convenient cloning method by the construction of an enrichment 
library has been developed for the isolation of trinucleotide repeats in human genome, 
and applied for the isolation of tri- and dinucleotide repeats in equine genome (10- 
14). The method includes the following procedures: adapter PCR of genomic DNA, 
enrichment procedure, adapter PCR for large preparation, followed by cloning. The 
enrichment procedure includes the following steps: capturing some other element from 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

81 



82 Tozaki 

genomic DNA by hybridization to biotin-labeled probes in solution, a nucleotide sub- 
strate-biased polymerase reaction, a subsequent extraction with magnetic beads coated 
with streptavidin. In particular, the nucleotide substrate-biased polymerase reaction is 
useful for the isolation of microsatellites containing long repeating units. The method 
is designed to select only the longer repeats. The advantage of the method is the effi- 
cient isolation of long repeat-containing highly polymorphic microsatellites, because 
microsatellites greater than 12-repeats in length are more likely to be polymorphic (75). 

In addition, this method offers the major advantage that it can be repeated any 
number of times for enrichment, indicating that the method is useful for the cloning of 
less frequent microsatellites, such as trinucleotide repeats. 

This chapter describes the construction of an enrichment library for the 
microsatellite-isolation. The method described here is a detailed version of a method 
that have been reported previously (10-14). The method is outlined in Fig. 1. 

2. Materials 

1. Autoclaved sterile 1.5-mL tube. 

2. Autoclaved sterile 200 uL-PCR tube. 

3. Purified genomic DNA. 

4. SauSAl (Takara Shuzo). 

5. 10X buffer for Sau3Al: 500 mM Tris-HCl, pH 7.5, at 25°C, 100 mM MgCl 2 , 10 mM 
dithiothreitol, 1000 mM NaCl. 

6. Mung bean nuclease (Takara Shuzo). 

7. IX buffer for Mung Bean nuclease: 30 mM acetate, pH 5.0, at 25°C, 100 mM NaCl, 1 mM 
zinc acetate, 5% glycerol. 

8. Autoclaved, distilled, deionized water. 

9. Oligonucleotide primers for adapter ligation. 
For first enrichment: 

Primer (1) 5'-GCACTCTCCAGCCTCTCAGTGCAG-3' (100 \iM and 25 \iM). 
Anti primer (1) 5'-GATCCTGCACTG-3' (100 uM). 
For second enrichment: 

Primer (2) 5'-AGCACTCTCCAGCCTCTCACCGAG-3' (100 \iM and 25 uM). 
Anti primer (2) 5'-GAT CCT CGG TGA-3' (100 uM). 

10. Biotin-labeled oligo (CAG) 8 (20 uAf). 

11. T4 DNA ligase (Takara Shuzo). 

12. 10X ligase buffer: 660 mM Tris-HCl, pH 7.6, at 25°C, 66 mM MgCl 2 , 100 mM 
dithiothreitol, 1 mM ATP. 

13. rTaq polymerase (5 U/uL) (Takara Shuzo). 

14. 10X rTaq buffer (MgCl 2 free): 100 mM Tris-HCl, pH 8.3, at 25°C, 500 mM KC1. 

15. ExTaq polymerase (5 U/uL) (Takara Shuzo). 

16. 1 OX ExTaq buffer (MgCl 2 plus) (Takara Shuzo) . 

17. LATaq polymerase (5 U/u,L) (Takara Shuzo). 

18. Perfect Match PCR enhancer (Stratagene, CA). 

19. An enzyme mixture: 0.5 (xL of rTaq polymerase, 0.2 u,L of 10X diluted LATaq poly- 
merase, and 2 u,L of Perfect Match PCR enhancer (see Note 5). 

20. dNTP (each 2.5 mM of dATP, dCTP, dGTP, and dTTP). 

21. d3NTP (each 2.5 mM of dATP, dCTP, and dGTP). 



Isolation of Trinucleotide Repeats 

1 



83 



T=H^ 



i' — r — I Step I 



1 adapter ligation and PCR amplification 
long repeat 



short repeat 



i — r 



i — r 



i — r 



^ denaturation 
1ZZZZZZZZZK. 



■&ZZZL 



' r 



i — r 



1 addition of biotinylated oligo 



i — r 



^F 



I 



3 



' — r 



I 



D 



i — r 



■ biotin trap using streptavidin 
y washing with high-stringency 

mmm 




y PCR amplification 



I 

Library construction 



Step II 



Step III 



Step IV 



nucleotide substrate 

biased polymerase reaction 
I I 



StepV 



Step VI 



Fig. 1. A schematic diagram of the construction of a library enriched for microsatellites. 
The first line represents the Sai<3AI-digested genomic DNA and the adapter sequences. The 
striped boxes are microsatellite target sequences, SA denotes the streptavidin-coated magnetic 
particles and B indicates biotin molecules. 



22. 
23. 
24. 
25. 
26. 



27. 



IX binding/washing buffer: 1 M NaCl, 10 mM Tris-HCl, pH 8.0, at 25°C, 1 mM EDTA. 
2X binding/washing buffer: 2 M NaCl, 10 mM Tris-HCl, pH 8.0, at 25°C, 1 mM EDTA. 
Alkaline buffer: 1 mM NaOH, 10 mM Tris, 1 mM EDTA. 
Coil DNA. 

Streptavidin MagneSphereR Paramagnetic Particle (Promega, WI). Wash by adding and 
removing 0.5X SSC at room temperature twice, and resuspend in 100 [xL of IX binding/ 
washing buffer with Cotl DNA (100 ng/(xL). 
Magna-Sep (a magnetic stand) (Gibco BRL, MD). 



84 Tozaki 

28. TE buffer: 10 rnM Tris-HCl, pH 8.0, at 25°C, 1 mM ethylenediaminetetraacetic acid 
(EDTA). 

29. Phenol equilibrated in TE buffer. 

30. Chloroform:isoamyl alcohol: 24:1, v/v. 

31. Glycogen (20 (xg/[xL) (Roche, Mannheim, Germany). 

32. 3 M Sodium acetate. 

33. 5 M Ammonium acetate. 

34. 100% Ethanol. 

35. 70% Ethanol. 

36. G-50 Spin column (Roche, Mannheim, Germany). 

37. T-vector (Promega, WI). 

38. Competent Escherichia coli (E. coli) cells for electroporation. 

3. Methods 

3. 1. Adapter PCR of Genomic DNA 

The steps described in this method correspond to step I and step II of Fig. 1. 

1. Add in the following order: approx 20 jig of genomic DNA, 10 [xL of 10X buffer for 
Sau3Al, 5 (xL of Saw3AI, and up to 100 [xL of sterile water. Incubate at 37°C overnight. 
Add 100 |xL of sterile water. 

2. Extract by adding 100 [xL of phenol that has been preequilibrated with TE buffer. Vortex. 
Add 100 [xL of chloroform:isoamyl alcohol. Vortex. Centrifuge in a microfuge at room 
temperature for 30 s to separate the phases. Remove the upper (aqueous) phase (approx 
200 (xL) and place in a fresh sterile 1.5-mL tube. 

3. Repeat step 2 once. 

4. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% 
ethanol. Incubate at -80°C for at least 10 min. 

5. Recover the restricted genomic DNA by centrifugation in a microfuge (12 OOOg) at 4°C 
for 20 min. Discard the supernatant and wash the pellet with 400 uE of cold 70% ethanol. 
Carefully discard the supernatant, remove excess liquid from the walls of the tube, and 
vacuum-dry the pellet in Speed- Vac for 5 min. Resuspend the pellet in 50 uE of TE buffer 
(see Note 1). 

6. In a sterile 1.5-mL tube, add in the following order: 500 ng of the restricted genomic 
DNA, each 2.5 [xL of 100 \iM primer (1) and antiprimer (1), 3 [xL of 10X buffer for ligase, 
and up to 30 uE of sterile water. 

7. Incubate at 53°C for 10 min in a heat block. 

8. Incubate the tube with the heat block in refrigerator at 4 C C for 60 min. 

9. Add 1 |xL of ligase, and incubate at 12 C C overnight. 

10. Add 470 uE of sterile water. 

11. In a sterile 200-u.L PCR tube, add in the following order: 10 |xL of the ligation mixture, 5 uE 
of 20 |xM primer (1), 10 [xL of 10X buffer for ExTaq polymerase (MgCl 2 plus), 8 (xL of 
each 2.5 raM dNTP, 0.5 uE of ExTaq polymerase, and 66.5 [xL of sterile water. 

12. Prepare four tubes for the sample. 

13. Amplify by PCR using the following cycle profiles: initial denaturation at 72°C for 5 min; 
approx 20 cycles of 1 min each at 94°C and 72°C; and then 10 min at 72°C for final 
extension (see Note 2). 

14. Extract by adding 200 [xL of phenol that has been preequilibrated with TE buffer. Vortex 
it. Add 200 [xL of chloroform:isoamyl alcohol. Vortex and centrifuge it in a microfuge at 



Isolation of Trinucleotide Repeats 85 

room temperature for 30 s to separate the phases. Remove the upper phase (approx 400 [xL) 
and place in a fresh sterile 1.5-mL tube. 

15. Repeat step 14 once. 

16. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% 
ethanol. Incubate at -80°C for at least 10 min. 

17. Recover the PCR products by centrifugation in a microfuge (12 OOOg) at 4 C C for 20 min. 
Discard the supernatant and wash the pellet with 600 [xL of cold 70% ethanol. Carefully 
discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry 
the pellet in Speed-Vac for 5 min. 

18. Resuspend the pellet in 50 |xL of IX mung bean nuclease buffer. 

19. Add 1 [xL of mung bean nuclease. Incubate at 37 C C for 45 min. Add 150 \iL of sterile water. 

20. Extract by adding 100 [xL of phenol that has been preequilibrated with TE buffer. Vortex. 
Add 100 (xL of chloroform:isoamyl alcohol. Vortex and centrifuge it in a microfuge at 
room temperature for 30 s to separate the phases. Remove the upper phase (approx 200 [xL) 
and place in a fresh sterile 1.5-mL tube. 

21. Repeat step 20 once. 

22. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% 
ethanol. Incubate at -80°C for at least 10 min. 

23. Recover the PCR products by centrifugation in a microfuge (12 OOOg) at 4 C C for 20 min. 
Discard the supernatant and wash the pellet with 600 (xL of cold 70% ethanol. Carefully 
discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry 
the pellet in Speed-Vac for 5 min. Resuspend the pellet in 50 (xL of TE buffer. 

24. Purify the PCR product by passing the remaining excess primers through a G-50 spin 
column by centrifugation (see Notes 1 and 3). 

3.2. Enrichment Procedure 

The steps described in this method correspond to step III, step IV, and step V of Fig. 1. 

1. In a sterile 200-[xL PCR tube, add in the following order: 500 ng of the purified PCR 
products, 1 u,L of 20 \xM biotinylated oligo-(CAG) 8 , 10 [xL of 10X buffer for tTaq 
polymerase (MgCl 2 free), 10 ^L of 15 mM MgCl 2 , 8 [xL of each 2.5 mM d3NTP, and up 
to 100 fxL of sterile water. As negative controls, prepare two reaction mixtures without 
the purified PCR products or the biotinylated oligo-(CAG) 8 , respectively. 

2. Denature the sample at 94°C for 3 min to make target microsatellites accessible to the 
probe, and incubate it for each 5 min at 80°C and 75 C C, respectively, to hybridize the 
biotinylated oligo-(CAG) 8 to the denatured PCR products. 

3. Add 2.7 u.L of enzyme mixture to the reaction solution kept at 75 C C above (see Note 4). 

4. Incubate it at 77°C for 10 min to carry out the nucleotide substrate-biased polymerase 
reaction. 

5. Chill on ice, then move all the solution to a fresh 1 .5-mL tube on ice which contains 200 (xL 
of 2X binding/washing buffer to stop the polymerase reaction. 

6. Add 100 uL of streptavidin-coated magnetic beads in the presence of 100 ng/u.L of Cotl 
DNA as the competitor. 

7. Incubate the sample at 37°C for 30 min (see Note 5). 

8. Capture the magnetic bead-complexes with a magnet stand, and then remove the superna- 
tant containing excess unbound oligos and noncomplementary sequences from the tube. 

9. Wash the sample by adding 500 (xL of IX binding/washing buffer at room temperature. 
Gently pipet the sample. Capture the magnetic bead-complexes with the magnet stand. 
Remove the supernatant. 



86 Tozaki 

10. Repeat step 9 twice. 

11. Wash by adding 100 [xL of IX binding/washing buffer at 80°C. Gently pipet the sample. 
Capture the magnetic bead-complexes with the magnet stand. Remove the supernatant. 

12. Repeat step 11 once. 

13. Elute ssDNAs, which were containing repeat sequences, by adding and incubating 50 (xL 
of alkaline buffer at 80°C from the biotinylated oligo-(CAG) 8 . Gently pipet the sample. 
Capture the magnetic beads with the magnet stand. Recover the supernatant to a fresh 
1.5-|xL tube. 

14. Repeat step 13 once. 

15. Precipitate the eluted ssDNAs by adding 0.5 [xL of glycogen (20 (xg/u.L), 40 u.L of 5 M 
ammonium acetate, vortex briefly, and then 400 [xL of cold 100% ethanol. Incubate at 
-80°C for at least 15 min. 

16. Recover the ssDNAs by centrifugation in a microfuge (12 OOOg) at 4°C for 20 min. Dis- 
card the supernatant and wash the pellet with 600 u,L of cold 70% ethanol. Carefully 
discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry 
the pellet in Speed-Vac for 5 min. Resuspend the pellet in 20 jxL of TE buffer. 

3.3. Adapter PCR for Large Preparation of the Repeat Sequences 

The step described in this method corresponds to step VI of Fig. 1. 

1 . In a sterile 200-fxL PCR tube, add in the following order: 2 (xL of the resuspended ssDNAs, 
2 u,L of 20 [xM primer (1), 5 jxL of 10X buffer for ExTaq polymerase (MgCl 2 plus), 4 u,L 
of each 2.5 mM dNTP, 0.25 fxL of ExTaq polymerase, and 36.75 (xL of sterile water. Also 
prepare reaction mixtures of the two resuspended products without the purified PCR prod- 
ucts or the biotinylated oligo-(CAG) g , respectively (see Note 6). 

2. Amplify by PCR using the following cycle profiles: initial denaturation at 94 C C for 2 min; 
15 cycles of 1 min each at 94 C C and 72 C C; and then 10 min at 72°C for final extension. 

3. As a preliminary experiment, confirm the number of cycles of the second PCR procedure 
for large preparation. In three fresh sterile 200-(xL PCR tubes, add in the following order: 
10 [xL of the first PCR products of sample, the negative control without the purified PCR 
products, or the negative control without the biotinylated oligo-(CAG) 8 as templates, 
4 (xL of 20 [xM primer (1), 10 [xL of 10X buffer for ExTaq polymerase (MgCln plus), 8 (xL 
of each 2.5 mM dNTP, 0.5 [xL of ExTaq polymerase, and 67.5 fxL of sterile water. 

4. Amplify the sample DNA by PCR using the following cycle profiles: initial denaturation 
at 94°C for 2 min; approx 13-18 cycles of 1 min each at 94°C and 72°C; and then 10 min 
at 72°C for final extension. 

5. Take 5-10 fxL aliquot of the PCR to visualize the products by agarose gel electrophoresis 
(1.5% agarose). Confirm the number of cycles of the second PCR that does not amplify 
PCR products of the two negative controls. 

6. After the confirmation, amplify the first PCR products by PCR using the same condition 
and the confirmed cycle profiles. 

7. Prepare and amplify four tubes of the PCR products with the condition of step 6. 

8. Extract by adding 200 (xL of phenol that has been preequilibrated with TE buffer. Vortex. 
Add 200 [xL of chloroform:isoamyl alcohol. Vortex. Centrifuge in a microfuge at room 
temperature for 30 s to separate the phases. Remove the upper phase (approx 400 [xL) and 
place in a fresh sterile 1.5-mL tube. 

9. Repeat step 9 once. 

10. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% 
ethanol. Incubate at -80°C for at least 10 min. 



Isolation of Trinucleotide Repeats 87 

11. Recover the PCR products by centrifugation in a microfuge (12 OOOg) at 4°C for 20 min. 
Discard the supernatant and wash the pellet with 600 |xL of cold 70% ethanol. Carefully 
discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry 
the pellet in Speed-Vac for 5 min. Resuspend the pellet in 50 [xL of TE buffer. 

12. Purify the PCR product by passing the remaining PCR reaction through a G-50 spin col- 
umn by centrifugation (see Note 1). 

3.4. Second Cycle for Enrichment 

The step described in this method corresponds to step I of Fig. 1. 

1. When the second enrichment procedure was desired, add in the following order: 5 [xg of 
the first enriched PCR products, 10 [xL of 10X buffer for Sau3Al, 5 [xL of Sau3Al, and 
up to 100 [xL of sterile water. Incubate at 37°C overnight (see Note 7). Add 100 u.L of 
sterile water. 

2. Extract by adding 100 [xL of phenol that has been preequilibrated with TE buffer. Vortex 
the sample. Add 100 [xL of chloroform:isoamyl alcohol. Vortex and centrifuge it in a 
microfuge at room temperature for 30 s to separate the phases. Remove the upper phase 
(approx 200 (xL) and place in a fresh sterile 1.5-mL tube. 

3. Repeat step 2 once. 

4. Add 0.1 volume of 3 M sodium acetate. Mix well, and add 2 volumes of cold 100% 
ethanol. Incubate at -80°C for at least 10 min. 

5. Recover the PCR products by centrifugation in a microfuge (12,000g) at 4 C C for 20 min. 
Discard the supernatant and wash the pellet with 400 [xL of cold 70% ethanol. Carefully 
discard the supernatant, remove excess liquid from the walls of the tube, and vacuum-dry 
the pellet in Speed-Vac for 5 min. Resuspend the pellet in 50 (xL of TE buffer. 

6. Purify the PCR product by passing the remaining PCR through a G-50 spin column by 
centrifugation. 

7. In a sterile 1.5-mL tube, add in the following order: 250 ng of the purified PCR products, 
each 2.5 fxL of 100 [xM primer (2) and antiprimer (2), 3 (xL of 10X buffer for ligase, and 
up to 30 [xL of sterile water. 

8. Incubate at 53°C for 10 min in the heat block. 

9. Incubate the tube with the heat block at 4°C for 60 min. 

10. Add 1 [xL of ligase, and incubate at 12°C overnight. 

1 1 . Add 70 u.L of sterile water. 

12. Repeat steps 3.1.3-3.1.5, 3.2, and 3.3 for second cycle enrichment. 

3.5. Construction of Enrichment Library 

The PCR products enriched for (CAG) n repeats should be constructed by direct 
cloning into a T-vector using T4 DNA ligase, taking advantage of the 3'-A overhangs 
often produced by Taq polymerase. These recombinants should be transformed into 
competent E. coli cells by electroporation (see Note 8). 

4. Notes 

1 . Take an aliquot containing 500 ng of genomic DNA or the PCR products to check the 
enrichment rate of microsatellites by Southern blot analysis (see Note 8). 

2. With the adapter PCR procedure, PCR conditions are optimized to generate a smear of 
the PCR products without specific bands. If some specific bands appear, the adapter 
sequences (primer 1 or primer 2) should change to new designed adapter sequences. The 



88 Tozaki 

enrichment rate of microsatellites would be influenced by the generation of specific bands. 
By increasing amount of the ligation mixtures, the number of cycles for PCR could be 
reduced. It is possible that the reduced cycles of the PCR might get rid of the specific 
bands by the adapter primers. 

3. Check the absence of excess primers by agarose gel electrophoresis. If the excess primers 
are visualized with the electrophoresis, the methods to purify them (see Subheading 
3.1.14-24) should be repeated until the absence of the excess primers. The presence of 
the excess primers decreases the efficiency of the nucleotide substrate-biased polymerase 
reaction. 

4. Prepare the fresh enzyme mixtures to prevent the inactivation of the enzymes. 

5. Gently mix the reaction tubes, because the magnetic beads precipitate during the 
incubation. 

6. The confirmation of the number of cycles of PCR for large preparation should be carried 
out for every lot of the experiments. The PCR with superfluous cycles might reduce the 
enrichment rate of microsatellites. 

7. For this method to be repeated, the primer for the second enrichment must be changed 
from that for the first enrichment. The adapter change prevented amplification from the 
first primer. Amplification with alternative sets of primers would then produce different 
specific libraries of sequences complementary to a common target. 

8. To analyze the enrichment rate, we performed Southern blot analysis of each product. 
The results reveal that the enrichment cloning method achieved 10 2 -fold enrichment by 
the first round procedure. Finally, 10 2_3 -fold enrichment was achieved for (CAG) n repeat 
by the second round. A potential risk of the second round of PCR was considered, 
because a single DNA fragment was amplified to produce many copies. The results 
obtained in the previous study, however, indicated that only about 10% of the clones 
isolated were identical (11). 

References 

1. Weber, J. L. and May, P. E. (1989) Abundant class of human DNA polymorphisms which 
can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44, 388-396. 

2. Litt, M. and Luty, J. A. (1989) A hypervariable microsatellite revealed by in vitro ampli- 
fication of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 
44,397-401. 

3. Tozaki, T., Sakagami, M, Mashima, S., Hirota, K., and Mukoyama, H. (1995) ECA-3: 
equine (CA) repeat polymorphism at chromosome 2pl.3-4. Anim. Genet. 26, 283. 

4. Marklund, S., Ellegren, H., Eriksson, S., Sandberg, K., and Andersson, L. (1994) Parent- 
age testing and linkage analysis in the horse using a set of highly polymorphic horse 
microsatellites. An™. Genet. 25, 19-23. 

5. Takahashi, H., Nirasawa, K., Nagamine, Y., Tsudzuki, M., and Yamamoto, Y. (1998) 
Genetic relationships among Japanese native breeds of chicken based on microsatellite 
DNA polymorphisms. ./. Hered. 89, 543-546. 

6. Gerber, H. .P., Seipel, K., Georgiev, O., Hofferer, M., Hug, M., Rusconi, S., and Schaffner, 
W. (1994) Transcriptional activation modulated by homopolymeric glutamine and proline 
stretches. Science 263, 808-811. 

7. The Huntington's Disease Collaborative Research Group (1993) A novel gene containing 
a trinucleotide repeat that is expanded and unstable on Huntington's Disease chromo- 
somes. Cell 72, 971-983. 



Isolation of Trinucleotide Repeats 89 

8. Orr, H. T., Chung, M. Y ., Banfi, S., Kwiatkowski, T. J. Jr., Servadio, A., Beaudet, A. L., 
et al. (1993) Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia 
type I.Nat. Genet. 4, 221-225. 

9. Gastier, J. M, Pulido, J. C, Sunden, S., Brody, T., Buetow, K. H., Murray, J. C, et al. 
(1995) Survey of trinucleotide repeats in the human genome: assessment of their utility as 
genetic markers. Hum. Mol. Genet. 4, 1829-1836. 

10. Inoue, S., Takahashi, T., and Ohta, M. (1999) Sequence analysis of genomic regions con- 
taining trinucleotide repeats isolated by a novel cloning method. Genomics 57, 169-172. 

11. Tozaki, T., Inoue, S., Mashima, S., Ohta, M., Miura, N., and Tomita, M. (2000) Sequence 
analysis of trinucleotide repeat microsatellite from an enrichment library of the equine 
genome. Genome 43, 354-365. 

12. Tozaki, T., Kakoi, H., Mashima, S., Hirota, K., Hasegawa, T., Ishida, N., et al. (2000) The 
isolation and characterization of 18 equine microsatellite loci, TKY272-TKY289. Anim. 
Genet. 31, 149-150. 

13. Tozaki, T., Kakoi, H., Mashima, S., Hirota, K., Hasegawa, T., Ishida, N., et al. (2000) The 
isolation and characterization of 36 equine microsatellite loci, TKY290-TKY323. Anim. 
Genet. 31, 234-236. 

14. Tozaki, T., Mashima, S., Hirota, K., Miura, N., Choi-Miura, N., and Tomita, M. (2001) 
Characterization of equine microsatellites and microsatellite-linked repetitive elements 
(eMLREs) by efficient cloning and genotyping method. DNA Res. 8, 33-45. 

15. Weissenbach, J., Gyapay, G., Dib, C, Vignal, A., Morissette, J., Millasseau, P., et al. 
(1992) A second-generation linkage map of the human genome. Nature 359, 794-801. 



10 

Methylation-Specific PCR 
Haruhiko Ohashi 



1. Introduction 

1.1. Significance of DNA Methylation as an Epigenetic Phenomenon 

Methylation of the DNA is an important epigenetic (i.e., not associated with alter- 
ation in the primary structure of the DNA) phenomenon, which plays important roles 
in regulation of gene expression, maintenance of genome integrity, and genomic 
imprinting. Although not only other nucleotides, but also proteins and lipids can be 
methylated, in the context of the present discussion, "methylation" designates only 
that of cytosine residues that are located 5' to guanines (CpG cytosines). A methyl 
residue is added to the 5 position of the pyrimidine ring of cytosine (5-methylcytosine) 
in the course of DNA replication, a process mediated by DNA(cytosine-5)- 
methyltransferases. 

1.2. Other Methods for DNA Methylation Analysis 

Investigation on DNA methylation had long been performed by Southern 
hybridization with methylation-sensitive endonucleases. Some endonuleases digest 
double-strand DNA at their cognitive sequences only when cytosines within are free 
of methylation, whereas others do so, regardless of the presence or absence of 
5-methylcytosines. Genomic DNA is digested by a methylation-sensitive endonuclease 
and electrophoresed on agarose gel and probed with specific DNA probe. If the cyto- 
sines in the endonuclease recognition site is methylated, the DNA is left uncleaved at 
that site, thus appears band(s) of larger-than-expected size(s). 

The main problems with Southern blotting with methylation-sensitive endonu- 
cleases is that only the cytosines in the context of available methylation-sensitive endo- 
nucleases [i.e., Hpall (CCGG), Hhal (GCGC)] could be examined; also, relatively 
large amount of DNA (5-10 u,g) is required for each analysis. The latter problem 
could be circumvented by the use of polymerase chain reaction (PCR). Since cytosine 
methylation should not be conserved after PCR (5-methylcytosines should be con- 
verted to cytosines), digestion by methylation-sensitive endonucleases should be per- 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

91 



92 Ohashi 

formed before amplification by polymerase chain reaction (PCR). Primers should be 
positioned upstream and downstream of the recognition site of a methylation-sensitive 
endonuclease. DNA digested by a methylation-sensitive endonuclease is amplified 
with the primers: If the restriction site is methylated, the template DNA should be left 
uncleaved, thus the PCR product should be generated; and if the site is unmethylated, 
because the template is cut into fragments, there should be no product. Even if the 
template DNA is fully unmethylated at the restriction site, however, only slight in- 
complete digestion may lead to amplification because of high sensitivity of PCR. Thus, 
interpretation of the results should be made with caution for this method. 

In 1992, Frommer et al. (1) described a method called bisulfite sequencing. This 
strategy of identifying the presence and absence of cytosine methylation has greatly 
contributed to the advancement of research on DNA methylation. Treatment of dena- 
tured DNA by sodium bisulfite deaminates cytosines at their 4 position, and converts 
cytosines to uracils. When the bisulfite-treated DNA is subjected to PCR, uracils are 
converted to thymines. 5-Methylcytosines are resistant to deamination by sodium 
bisulfite, and converted to cytosines after PCR. Thus, after PCR following bisulfite 
treatment, unmethylated cytocines are converted to thymines, while methylated cyto- 
sines are changed to cytosines (see Fig. 1). This means that cytosine methylation, 
which is an epigenetic modification of the DNA, can be translated into difference in 
primary structure (base composition) of the DNA. In bisulfite sequncing, the ampli- 
fied products are cloned and sequenced. Because all the non-CpG cytosines are 
uniformly unmethylated, only the 5-methylcytosines in CpGs should remain cytosines 
on sequencing. Comparison with the original DNA sequence shows which cytosines 
are methylated. 

1.3. Basic Concept of Methylation-Specific PCR 

Methylation-specific PCR (MSP), which was first described by Herman et al. (2) in 
1996, is an application of bisulfite sequencing method. For a sequence in a gene 
containing CpGs, the allele on which those CpGs are methylated and another on which 
those CpGs are unmethylated should give different sequences after bisulfite modifica- 
tion. When a primer set that are complementary to the sequence with methylated CpGs, 
but are not complementary to the originally same sequence with unmethylated CpGs, 
is used for PCR, only the sequence (allele) with methylated CpGs should be amplified. 
The same is true for the primer pair specific for sequence with unmethylated CpGs. 
The interpretation of the result is simple: If PCR product of the expected size is seen 
on agarose gel electrophoresis, the sample is considered to contain the methylated or 
unmethylated allele of the gene, depending on the primer pair used. Usually, primer 
pairs specific for methylated and unmethylated sequences, respectively, are used 
for the same gene, and the amplified products are run side-by-side on agarose gel for 
comparison. 

1.4. Application of MSP 

The greatest advantage of MSP over other methods of DNA methylation analysis is 
its simplicity. In contrast to Southern blotting with methylation-sensitive restriction 
enzymes, MSP requires much less amount of DNA, isotope use is usually unneces- 



Methylation-Specific PCR 



93 




CHs 



Fig. 1. Conversion of pyrimidines residues by chemical and biochemical reactions. Changes 
between pyrimidine residues by methylation, deamination, and PCR are being shown. Cytosines 
in DNA strands can be methylated to be 5-methylcytosines by DNA(cytosine-5)- 
methyltrasnferases in vivo, or by other methylases in vitro. Sodium bisulfite deaminates cy- 
tosines to uracils, whereas 5-methylcytosines are resistant to modification. PCR with dCTP as 
nucleotide source converts 5-methylcytosines to cytosines and uracils to thymines, respectively. 



sary, and any CpGs, regardless of the sequence around, can be evaluated. Also, inter- 
pretation of the results is much more straightforward. MSP is superior to bisulfite 
sequencing in that it does not require cloning and sequencing, which usually take sev- 
eral days, and can be done in 1 or 2 d. All these characteristics fit well to researches 
that examine a large number of clinical samples at a time, and indeed it has been 
widely used for detection of aberrant hypermethylation and inactivation of tumor sup- 
pressor genes in cell lines and tumor samples (3). This method can also be used to 
evaluate methylation status of any DNA sequences, such as viral genes (4), and 
imprinted X-linked and autosomal genes (5 and 6). 

2. Materials 

2. 1. Bisulfite Modification of the DNA 

1. Hydroquinone (Sigma, St. Louis, MO). 

2. Sodium bisulfite (Sigma). 

3. Wizard DNA purification resin (Promega, Madison, WI), or similar product. 

4. CpGenome DNA Modification Kit (Intergen, Purchase, NY). 

5. 3 M Sodium hydroxide (NaOH). 



94 Ohashi 

6. 70 % Ethanol. 

7. TE Buffer: 10 mM Tris-HCl, 0.1 mM ethylenediamine tetraacetic acid (EDTA), pH 7.5. 

8. 5 M Ammonium acetate (NH 4 OA c ). 

9. 5 M Sodium chloride (NaCl). 

2.2. PCR with Methylation-Specific Primers 

1. 10X PCR reaction buffer: 100 mM Tris-HCl (pH 8.3), 500 mM KC1, 15 mM MgCl 2 , 
(TaKaRa, Osaka, Japan). 

2. dNTP mixture: 2.5 mM each (TaKaRa). 

3. Taq DNA polymerase (TaKaRa). 

3. Methods 

The procedure of the MSP consists of two steps: bisulfite modification of the sample 
DNA, and PCR with methylation-specific primers. Chemical modification of double- 
strand DNA by sodium bisulfite consists of four steps: 

1. Denaturation of the double-strand DNA by NaOH (see Note 1); 

2. Sulfonation of the 6 position of cytosine by sodium bisulfite (cytosine sulfonate); 

3. Hydrolytic deamination at 3 position by hydroquinone (uracil sulfonate); 

4. Alkali desulfonation by NaOH (uracil). 

These reactions can be carried out with the protocol described below. Alternatively, 
a kit for bisulfite modification (CpGenome DNA Modification Kit, Intergen) is com- 
mercially available. It is important to note that each single-strand DNA (the sense 
strand and the antisense strand), generated as the result of initial denaturation, is modi- 
fied independently by sodium bisulfite in the subsequent steps. This means that the 
sense and antisense strands are no longer complementary to each other after modifica- 
tion. Bisulfite-modified DNA can be stored at -20°C and used for repeated analyses. 

PCR with methylation-specific primers is not different from ordinary PCR, but 
needs some precautions (see Note 2). There may be three sets of methylation-specific 
primers for each gene, or a region of a gene: primers that amplify sequences on which 
CpGs are methylated (M-primers); primers that amplify sequences on which CpGs are 
unmethylated (U-primers); and primers that are designed for the region without CpGs 
and should amplify the gene regardless of methylation status (C-primers, C for com- 
mon). M- and U-primers are the ones used for MSP, and C-primers are for bisulfite 
sequencing. There could be another set of primers, W-primers (W for wild-type), which 
amplify DNA not modified by sodium biulfite, or escaped modification for some tech- 
nical defects. PCR with the forward M-primer and the reverse M-primer should detect, 
if present, the gene that are methylated at these CpGs. PCR with the forward M-primer 
and the reverse C-primer, or the other way around, should also work for the purpose. 
Because selecting regions fit for M- and U-primers is sometimes difficult, while the 
region without CpGs can usually be found, combination of either the forward M-primer 
or the forward U-primer, and the reverse C-primer may be a practical choice. 

In fact, it is fairly easy to perform MSP on a gene for which workable methylation- 
specific primer sequences are already reported. If one wishes to examine a gene for 
which MSP has not been performed by others, the difficult part is selecting methyla- 
tion-specific primers. The primers used in MSP should be designed so that they dis- 



Methylation-Specific PCR 95 

M-primer: 5'-CG AGCGTAGTATTT TTCGGC-3' 

WT; 5'-ACCCAGAGGCCGCGAGCGCAGCACCTCCCGGCGCCAGT -3' 
U-primer: 5'-GGTTGr GAGTO TAGTATTT TTTGGI-3 ' 

Fig. 2. Example of primer selection for MSP. MSP primers for the human androgen receptor 
gene (HUMARA) is being shown (5), as an example of primer designing. WT: Unmodified 
sense strand sequence rich in CpG and non-CpG cytosines. M-primer: Primer specific for the 
methylated allele. All the cytosines except for those preceding guanines are changed to thym- 
ines, while cytosines 5' to guanines remain unchanged. The Ts are thymines converted from 
cytosines. The Cs are CpG cytosines that are methylated and are not to be converted to thym- 
ines. U-primer: Primer specific for the unmethylated allele. All the cytosines are changed to 
thymines. The Ts are thymines converted from cytosines. The 7s are CpG cytosines that are 
unmethylated and are to be converted to thymines. 



criminate between methylated and unmethylated sequences and, at the same time, 
between bisulfite-modified and biulfite-unmodified sequences. Thus, the primers 
should be designed for the region rich in both CpGs and non-CpG cytosines. Both 
U- and M-primers must contain Ts that are non-CpG Cs in the unmodified (wild-type) 
sequence. U-primers must have Ts located 5' to Gs, at their 3' ends, and M-primers 
must contain Cs in the CpG context at their 3' ends. Fig. 2 shows, as an example, the 
genomic sequence and the methylation-specific primers designed for the human 
androgen receptor gene (5). If the purpose of the investigation is to know whether the 
expression of a gene is regulated by methylation, the part of the gene to be examined is 
the promoter region of the gene, where clustering of CpGs (CpG islands) is found 
about half of the genes. Sometimes, however, the CpG island in the 5' region of a gene 
may extend to the 5' untranslated region (5'-UTR) or even to the coding region. 

3. 1. Bisulfite Modification of the DNA 

1. Take 1 u.g of DNA and add double-distilled water to 50 (xL in a 1.5-mL tube. 

2. Add 3.5 uE of 3 M NaOH (final concentration of 0.2 M), and incubate at 37°C or 10 min. 

3. Freshly prepare 10 mM hydroquinone, and add 35uL of it to the tube. 

4. Freshly prepare 3 M sodium bisulfite, adjust to pH 5.0 by adding 3 M NaOH, add 520 u,L 
of it to the tube, mix well, overlay with mineral oil, and incubate at 50 C C for 16 h or 
longer. 

5. Put the tube on ice, add 5 uE of Wizard DNA purification resin (or similar DNA purifica- 
tion resin), mix, and incubate at room temperature for 10 min, spin at 5000g for 10 s at 
room temperature, discard the supernatant. 

6. Put 1 mL of 70% ethanol, mix by Vortex, spin at 5000g for 10 s, and discard the superna- 
tant. Do this two more times, and remove the supernatant completely. 

7. Add 50 uE of TE, mix well, incubate at 50°C for 5 min, spin at 12,000g for 1 min, and 
transfer the supernatant to a fresh 1.5-mL tube. 

8. Add 5.5 uL of 3 M NaOH, mix, and incubate at room temperature for 5 min. 

9. Add 10 u.L of 5 M NH 4 OAc and mix (for neutralization). 

10. Add 1 uL of 5 M NaCl and 200 uE of 100% ethanol, mix well, keep the tube at -20°C for 
1 h. Spin at 12 OOOg, at 4°C for 5 min, discard the supernatant, rinse the pellet with 70% 
ethanol, dry the pellet, and dissolve in 20-50 uE of TE, and store at -20°C. 



96 Ohashi 

3.2. PCR with Methylation-Specific Primers 

1 . Make mixture of the following for the number of samples to be examined, and put the 
mixture into a 0.5-mL tube for PCR (see Note 3). 

a. 10X PCR reaction buffer (15 mM Mg 2+ ) 2 ixL; 

b. 2.5 mM dNTP mixture 2 [xL; 

c. forward primer (10 \xM) 1 [xL; 

d. reverse primer (10 \iM) 1 fxL; 

e. ddH 2 10 \xL. 
Overlay with mineral oil. 

2. Add the bisulfite-modified DNA (2 u,L), or a control sample (see Note 4) into the tube. 

3. Put the tubes on a thermal cycler and start denaturation at 95 C C for 5 min. 

4. Make mixture of the following for the number of tubes. 

a. Tag DNA polymerase (5 U/[iL) 0.2 u,L; 

b. ddH20 1.8 ixL. 

Take a tube from the thermal cycler, add 2 u,L of the Tag mixture to each tube through 
mineral oil, and put the tube back to the heat block. This should be done as quick as 
possible. 

5. Go through 30-40 cycles of the following amplification (see Note 3). 

a. Denaturation 95 C C for 30 s; 

b. Annealing 55°C for 30 s; 

c. Extension 72°C for 30 s. 

6. Take 5-10 (xL of the reaction solution and run on 2-3% agarose gel. 

4. Notes 

1. Complete denaturation of the DNA in the first step of bisulfite modification is important, 
because unmethylated cytosines in undenatured DNA cannot be converted to uracils, thus 
leads to misinterpretation that they are methylated. For bisufite sequencing of specific 
genes or plasmids, digestion of the template DNA by a endonuclease that cleaves outside 
the region to be amplified is recomended, in order to ensure complete denaturation (1). 
This pretreatment of DNA does not fit to examination for clinical samples, with which 
more than one genes may be examined for methylation. Another strategy for ensuring 
initial denaturation is to shear DNA through fine gage needles. Our experience with MSP 
for various genes without such pretreatment, however, did not show inconsistent results, 
and it seems that pretreatments may not be necessary. One can always check, if needed, 
the validity of bisulfite treatment of the given gene by PCR with W-primers or by sequenc- 
ing MSP product. 

2. By experience amplification efficiency with MSP is lower than with ordinary PCR, and 
one often needs to optimize PCR condition, in the direction of lowering stringency, to see 
clearer bands. The situation sometimes becomes tricky, because the M-primers and 
U-primers are designed to recognize sequences only partially different to each other. 
When one lowers the stringency of PCR and undergo a larger number of cycles to get 
clearer bands, they may notice that specificity of the reaction is lost: the same band can be 
seen with the control samples with the opposite specificity (e.g., M controls for U-prim- 
ers). This may be the greatest pitfall for methylation analysis with MSP. 

3. The factors that appear to influence PCR efficiency are reaction buffer [Mg 2+ concentra- 
tion, addition of demethyl sulfoxide (DMSO)], annealing temperature, and hot start regi- 
mens. We usually start with commercially available 10X PCR reaction buffer (10 mM 



Methylation-Specific PCR 97 

Tris-HCl (pH 8.3), 50 vaM KC1, 1.5 voM MgCl 2 , final concentration), annealing tempera- 
ture of 55°C, and hot start by adding Taq polymerase after incubating the reaction for 
5 min at 95°C. When no product of the expected size is seen after agarose electrophoresis, 
elevation of MgCli concentration, addition of DMSO (5% of the volume) should be tried. 
Elevating the annealing temperature may also help. Hot start appears to be critical for 
MSP, thus we usually do not even try nonhot start protocols. When ordinary hot start 
protocol of adding Taq after incubation at 95°C does not work, use of reagents designed 
for hot start, such as Platinum Taq (Gibco-BRL, Rockville, MD) may still help. Optimi- 
zation of PCR conditions for MSP seems much more difficult than for ordinary PCR, and 
it may sometimes be wiser to redesign primer sequences before comparing all the pos- 
sible conditions. 
4. Not only for initial optimization of MSP but also for each experiment, as for any PCR, 
control templates are necessary. To obtain the perfect controls for MSP is sometimes 
difficult, since one needs both DNA that is methylated at the CpGs of the gene of interest, 
and DNA that is unmethylated in the same region. The ideal controls for U-primers are 
the PCR-amplified DNA segment, because PCR products are fully unmethylated. If one 
methylates the PCR product with methylases in vitro, that would be a good control for 
M-primers. There are several kinds of commercially available bacterial methylases with 
different sequence specificity. For example, Sss I methylase (New England Biolabs, 
Beverly, MA) methylates all the CpG cytosines. All these preparations are time consum- 
ing, but may be necessary for analysis of nonhuman (i.e., viral) genes. When DNA from 
some sources (e.g., cell lines) in which the gene in question is known to be either methy- 
lated or unmethylated is available, that could serve as controls. When one possesses DNA 
that is unmethylated for the gene, then in vitro treatment with methylases can provide 
controls for methylated gene. 

References 

1. Frommer, M., McDonald, L. E., Miller, D. S., Collis, C. M., Watt, F., Grigg, G. W„ 
Molloy, P. L., and Paul, C. L. (1992) A genomic sequencing protocol that yields a positive 
display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. 
USA%9, 1827-1831. 

2. Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D., and Baylin, S. B. (1996) Methy- 
lation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc. Natl. 
Acad. Sci. USA 93, 9821-9826. 

3. Herman, J. G., Civin, C. I., Issa, J. P., Collector, M. I., Sharkis, S. J., and Baylin, S. B. 
(1997) Distinct patterns of inactivation of pl5INK4B and pl6INK4A characterize the 
major types of hematological malignancies. Cancer Res. 57, 837-841. 

4. Tao, Q., Swinnen, L. J., Yang, J., Strivastava, G., Robertson, K. D., Ambinder, R. F. 
(1999) Methylation status of the Epstein-Barr virus major latent promoter C in iatrogenic 
B cell lymphoproliferative disease. Application of PCR-based analysis. Am. J. Pathol. 
155,619-625. 

5. Uchida, T., Ohashi, H., Aoki, E., Nakahara, Y., Hotta, T., Murate, T., Saito, H., and 
Kinoshita, T. (2000) Clonality analysis by methylation-specific PCR for the human andro- 
gen-receptor gene (HUMARA-MSP). Leukemia 14, 207-212. 

6. Kubota, T., Das, S., Christian, S. L., Baylin, S. B., Herman, J. G., Ledbetter, D. H. (1997) 
Methylation-specific PCR simplifies imprinting analysis. Nature Genet. 16, 16-17. 



11 



Direct Cloning of Full-Length Cell 

Differentially Expressed Genes by Multiple Rounds 

of Subtractive Hybridization Based 

on Long-Distance PCR and Magnetic Beads 

Xin Huang, Zhenglong Yuan, and Xuetao Cao 
1. Introduction 

Subtractive cloning is an ideal technique for identifying genes differentially 
expressed in two nuclear acids population (1). The polymerase chain reaction (PCR)- 
based subtraction is the method of choice when the starting samples are heterogeneous 
or difficult to obtain, which often occurs in the tissues to be compared. PCR amplifica- 
tion is the easiest method for generating adequate amount of nuclear acids for mul- 
tiple-round hybridization. However, the bias in the relative representation of mRNA 
molecules in the starting materials and the accumulation of shorter fragments become 
the major deficiencies for this method and should be overcome. The bias caused by 
PCR amplification is because of the tendency of preferentially amplifying short frag- 
ments and certain templates with unique sequences in the sample. The thermophilic 
polymerase that is optimized to amplify multiple genes would be helpful and the adop- 
tion of gel filtration in preparation of templates for amplification could hinder the 
tendency of short fragment accumulation. In addition, increasing the amount of start- 
ing samples would represent much more molecules in tissues. 

For cloning differentially expressed genes with full-length, the tracer (from which 
to isolate differentially expressed sequences) is expected to generate full-length 
cDNAs. A reverse transcription (RT) method based on switching mechanism at 5' end 
of RNA template (SMART) can generate higher percentage of full-length cDNAs than 
that produced by conventional method (2,3). Amplifying the cDNAs by long-distance 
PCR (LD-PCR) (4) could improve the size of the product span and thus increase the 
proportion of full-length sequences. The requirement for the driver (which is used to 
hybridize tracer to remove common sequences) is different to that of the tracer: short 
fragments are better than long fragments because they have high mobility and could 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

99 



100 Huang, Yuan, and Cao 

approach tracer DNA easily in hybridization. In such circumstances, the limitation of 
PCR that small fragments are preferentially amplified is no longer a limitation. 

The hybridization step is very important to the success of the subtractive cloning 
and the tracendriver ratio is an important parameter controlling the efficiency of 
hybridization. The ratio should be at least 1:10 to enable the driver to govern the 
hybridization and much higher ratio would be better. We recommend the tracendriver 
ratio be at least 1:50 to 1:100. It is also important to remove driver-tracer hybrids 
and excess driver completely. Driver-tracer hybrids and excess driver could be 
removed efficiently by using streptavidin paramagnetic beads if the driver is 
biotinylated because streptavidin can bind biotin with high affinity (K d = 10~ 12 ) and 
biotin-streptavidin system had been found to be the most competent method for 
subtractive and positive selection (5,6). 

Taken together, to improve the efficiency of subtractive cloning and make the tech- 
nique less laborious, some preeminent methods were adopted to design a convenient, 
high-efficiency strategy for cloning of differentially expressed genes with full-length 
directly. First, a SMART method was employed, which not only can increase the popu- 
lation of full-length cDNAs, it can ligate the cDNAs with primers at the 5' and 3' 
termini as well when mRNA is being reverse transcribed. Second, the LD-PCR was 
adopted for the amplification of full-length cDNA. Third, to separate the tracer-driver 
hybrids and excess driver efficiently, the biotin-streptavidin system was used. Finally, 
to reduce the accumulation of shorter fragments, a Sepharcryl S-400 spin column chro- 
matography was very convenient and suitable. 

2. Materials 

2.1. Isolation of mRNA from Cells 

mRNA purification kit (Promega, Madison, WI, cat. no. MZ5400) including the 
following. 

1. GTC extraction buffer: 4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.1. 

2. 48.7 % [3-mercaptoethanol. 

3. Dilution buffer: 22.5 mM NaCl, 11.25 mM sodium citrate, 10 mM Tris-HCl, pH 7.4, 
1 mM ethylenediaminetetraacetic acid (EDTA), 0.25 % sodium dodecyl sulfate (SDS). 

4. 50 (xM Biotinylated Oligo(dT) probe. 

5. Streptavidin MagneSphere Paramagnetic Particles (SA-PMPs). 

6. 0.5X SSC: 75 mM NaCl, 37.5 mM sodium citrate. 

7. Nuclease-free water. 

8. 3 M NaAc, nuclease-free. 

9. Isopropanol. 
10. 70% ethanol. 

2.2. Preparation of Tracer 

1. 5X first-strand buffer: 250 mM Tris-HCl, pH 8.3, 375 mM KC1, 30 mM MgCl 2 , 50 mM 
dithiothreitol (DTT). 

2. lOmMdNTP. 

3. rRNasin (40 U/uL ) (Promega, Madison, WI, cat. no. N251 1). 

4. Superscript II RT (200 U/uL) (Gibco-BRL, Gaithersburg, MD, cat. no. 18064-014). 



Full- Length Subtractive Cloning 101 

5. 3' Anchoring primer: 5' TACGGCTGCGAGAAGACGACAGAAT (30) VN-3'. 

6. CAP-oligo: 5' TACGGCTGCGAGAAGACGACAGAAGGG-3' (Clontech, Palo Alto, CA). 

7. Tracer primer: 5' TACGGCTGCGAGAAGACGACAGAA-3'. 

8. 50X Advantage cDNA polymerase mix (Clontech, cat. no. 8417-1). 

9. 10X PCR buffer: 400 mM Tricine-KOH pH 9.2, 150 mM KAc, 35 mM Mg(Ac),, 
37.5 |ig/mL BSA. 

2.3. Preparation of Driver 

1. Avian myeloblastosis virus reverse transcriptase (AMV RT), 9 U/fxL (Promega, cat. 
no. M5101). 

2. 5X AMV RT buffer: 250 mM Tris-HCl, pH 8.3, 250 mM KC1, 50 mM MgCl 2 , 50 mM 
DTT, 2.5 mM spermidine. 

3. 40 mM sodium pyrophosphate. 

4. Recombinant Taq polymerase (any brand). 

5. 100|iMOligo(dT) 15 . 

6. 3' anchoring primer: 5' T (15 )(A/G/C)-3'. 

7. Biotin-21-dUTP (Clonetech, cat. no. 5021). 

8. 10X PCR buffer: 100 mM Tris-HCl, pH 9.0, 500 mM KC1, 15 mM MgCl 2 , 1% 
Triton X- 100. 

2.4. Hybridization 

1. 20X SSC: 3 M NaCl, 1.5 M sodium citrate. 

2. 10% SDS. 

3. Sephacryl S-400 HR (Amersham Pharmacia Biotech, Uppsala, Sweden, cat. no. 
17-0609-10). 

4. Spin column (Promega, cat. no. C128a). 

5. TEN: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 25 mM NaCl. 

6. SA-PMPs (Promega, cat. no. Z5482). 

7. PCR primer: 5' TACGGCTGCGAGAAGACGACAGAA-3'. 

8. 50X Advantage cDNA polymerase mix (Clontech, cat. no. 8417-1). 

9. 10X PCR buffer: 400 mM Tricine-KOH, pH 9.2, 150 mM KAc, 35 mM Mg(Ac) 2 , 
37.5 ixg/mL BSA. 

2.5. Cloning of Differentially Expressed Genes 

1. AmpliTaq Gold™ (Perkin-Elmer, Foster City, CA, cat. no. N808-0241). 

2. GeneAmp 10X PCR buffer: 100 mM Tris-HCl, pH 8.3, 500 mM KC1. 

3. 25mMMgCl 2 . 

4. pGEM-T vector systems (Promega, cat. no. A3600). 

5. Isopropylthio-|3-D-galactoside (IPTG). 

6. 5-bromo-4-chloro-3-indolyl-(5-D-galactoside (X-Gal). 

7. DH5ct Escherichia coli (E. coli strain). 

8. 0.1MCaCl 2 . 

9. Restriction endonucleases (New England Biolabs, Beverly, MA). 

10. Sequencing primers: SP6, 5' TCAAGCTATGCATCCAAC-3'; T7, 5' TCACTATAGGGC 
GAATTG-3'. 

11. BigDye terminator ready reaction mix (Perkin-Elmer, cat. no. 4303 154). 

12. 95% ethanol. 

13. Sequencing loading buffer. 



102 Huang, Yuan, and Cao 

2.6. Evaluation of the Efficiency of Subtraction 

1. Hybond-N™ Nylon membrane (Amersham, Little Chalfont, Buckinghamshire, UK, 
cat. no. RPN. 303N.) 

2. BluGene® Nonradioactive Nucleic Acid Detection System, which contains: 

a. SA-AP conjugate (1 mg/mL in 3 M NaCl, 1 mM MgCl 2 , 0.1 mM ZnCl 2 , 30 mM 
triethanolamine pH 7.6 ) 

b. Nitroblue tetrazolium (NBT) (75 mg/mL in 70% dimethylformamide). 

c. 5-bromo-4-chloro-3-indolylphosphate (BCIP) (50 mg/mL in dimethyl-formamide). 

3. Formamide. 

4. 20X SSC: 3 M NaCl, 1.5 M Sodium Citrate. 

5. 50X Denhardt's solution: 1% Ficoll 400, 1 % polyvinylpyrrolidone, 1% albumin bovine 
fraction V. 

6. 0.5 M sodium phosphate (pH 6.5): 65.25 g NaH 2 P0 4 -2H 2 0/L, 29.2 g Na 2 HP0 4 -12H 2 0/L. 

7. Shared herring sperm DNA. 

8. Dextran sulfate. 

9. Prehybridization solution: 50% formamide, 5X SSC, 5X Denhardt's solution, 20 mM 
sodium phosphate pH 6.5, 0.5 mg/mL freshly denatured sheared herring sperm DNA. 

10. Hybridization solution: 45% formamide, 5X SSC, IX Denhardt's solution, 20 mM sodium 
phosphate pH 6.5, 0.2 mg/mL freshly denatured sheared herring sperm DNA, 5% dextran 
sulfate. 

11. 7.5MNH 4 Ac. 

12. 5%SDS. 

13. Hybritube™ (Gibco-BRL, Cat. 20116-018, 10117-018). 

14. Buffer 1: 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl. 

15. Buffer 2: 3 % albumin bovine fraction V, 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl. 

16. Buffer 3: 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 50 mM MgCl 2 . 

17. Stopping solution: 20 mM Tris-HCl, pH 7.5, 0.5 mM Na 2 EDTA. 

3. Methods (see Note 1) 

3. 1. Isolation of mRNA from Cells 

Messenger RNA was isolated directly from the crude cells with the PolyATtract® 
System 1000 (Promega). About 1 x 10 6 to 1 x 10 7 peripheral blood mononuclear cells 
(PBMCs) or PHA stimulated PBMCs were harvested for mRNA purification each time 
(see Note 2). 

1. Wash cells with PBS twice and collect cells in a microcentrifuge tube. 

2. Add 400 uL of GTC extraction buffer into the microcentrifuge tube and 33 uL of p-mer- 
captoethanol (48.7%). Homogenize the cells completely with a small homogenizer. 

3. Add 800 uL of dilution buffer and 1.2 uL of Biotinylated Oligo(dT) Probe (50 \iM) and 
mix well. Incubate this mixture at 70°C for 5 min. 

4. Centrifuge this mixture at 12,000g for 10 min at room temperature and collect the 
supernatant. 

5. Wash 1 mL of SA-PMPs twice with equal volume of 0.5X SSC and suspend in 1 mL of 
0.5X SSC. Mix the SA-PMPs with the supernatant from step 4 and incubate at room 
temperature for 2 min. 

6. Wash the SA-PMPs with 0.5X SSC for three times by magnetic capturing. 



Full- Length Subtractive Cloning 103 

7. Add 200 uE of nuclease-free water to suspend the SA-PMPs completely and incubate at 
room temperature for 2 min. 

8. Collect the supernatant and add 20 uE of 3 M NaAc, 220 uE of isopropanol. Mix the 
contents well and incubate at -70°C overnight. 

9. Centrifuge the mixture at 15,000g for 15 min at 4°C. 

10. Add 1 mL of 70% ethanol and centrifuge at 15,000g for 5 min at 4°C. 

11. Dry the pellet briefly and elute it with nuclease-free water. Store at -70°C for later use. 

3.2. Preparation of Tracer 

3.2.1. First-Strand cDNA Synthesis 

The first strand cDNA was synthesized in a volume of 20 [iL (see Note 3). 

1 . Elute about 1 |xg of mRNA in 1 1 uE of nuclease-free water and add 4 uE of 5X first-strand 
buffer, 1 uE of 10 mM dNTP, 1 uE of rRNasin (40 U/uE), 1 uE of 3' Anchoring primer 
(10 u.M), 1 uE of CAP-oligo (10 uM), 1 uE of Superscript II RT (200 U/uE). 

2. Mix the contents well and incubate at 42°C for 1 h. 

3. Inactivate the reverse transcriptase by heating at 75°C for 15 min. 

3.2.2. Amplification of Tracer 

Advantage cDNA polymerase mix, which includes KlenTaq-1 polymerase and a 
minor quantity of Deep Vent polymerase, was used to amplify tracer in order to 
improve the proportion of full-length sequences (see Note 4). 

1 . 5 u.L of synthesized cDNA was used as template and mixed with 1 u.L of Advantage 
cDN A polymerase mix, 5 uE of reaction buffer, 1 nmole of dNTP and 20 pmole of tracer 
primer (see Subheading 2.2.). Add water to a total volume of 50 uE. 

2. PCR condition: 95°C for 1 min; 95°C for 10 s, 68°C for 4 min, repeat for 15 cycles; 68°C 
for 10 min (see Note 5). 

3.3. Preparation of Driver 

3.3. 1. First-Strand cDNA Synthesis (see Note 6) 

The first-strand cDNA of driver was synthesized by using AMV reverse tran- 
scriptase according to the routine method (7) and the manufacturer's instruction 
manual. 

1. Elute about 1 (xg of mRNA in 10 uE of nuclease-free water and mix with 4 uE of 5X 
AMV RT reaction buffer, 1 uE of 10 mM dNTP, 2 uE of 40 mM sodium pyrophosphate, 
1 uE of 100 \iM oligo(dT), 2 uE of AMV RT. Mix well and incubate at 42°C for 1 h. 

2. Inactivate AMV RT by boiling the reaction for 3 min. 

3.3.2. Preparation of Driver Labeled with Biotin 

Single-strand driver was amplified by primering with single primer and labeled 
with biotin-21-dUTP simultaneously. In 100 p,L PCR reaction volume, add 40 nmole 
of dNTP, 2 nmole of Biotin-21-dUTP, 100 pmole of 3' anchoring primer (see Sub- 
heading 2.3.), 2 \iL of synthesized cDNA, 20 U of Taq polymerase, 10 [iL of 10X 
PCR buffer. PCR condition: 95°C for 10 s, 42°C for 2 min, 72 C for 1 min and repeat 
for 40 cycles. 



Full- Length Subtractive Cloning 103 

7. Add 200 uE of nuclease-free water to suspend the SA-PMPs completely and incubate at 
room temperature for 2 min. 

8. Collect the supernatant and add 20 uE of 3 M NaAc, 220 uE of isopropanol. Mix the 
contents well and incubate at -70°C overnight. 

9. Centrifuge the mixture at 15,000g for 15 min at 4°C. 

10. Add 1 mL of 70% ethanol and centrifuge at 15,000g for 5 min at 4°C. 

11. Dry the pellet briefly and elute it with nuclease-free water. Store at -70°C for later use. 

3.2. Preparation of Tracer 

3.2.1. First-Strand cDNA Synthesis 

The first strand cDNA was synthesized in a volume of 20 [iL (see Note 3). 

1 . Elute about 1 |xg of mRNA in 1 1 uE of nuclease-free water and add 4 uE of 5X first-strand 
buffer, 1 uE of 10 mM dNTP, 1 uE of rRNasin (40 U/uE), 1 uE of 3' Anchoring primer 
(10 u.M), 1 uE of CAP-oligo (10 uM), 1 uE of Superscript II RT (200 U/uE). 

2. Mix the contents well and incubate at 42°C for 1 h. 

3. Inactivate the reverse transcriptase by heating at 75°C for 15 min. 

3.2.2. Amplification of Tracer 

Advantage cDNA polymerase mix, which includes KlenTaq-1 polymerase and a 
minor quantity of Deep Vent polymerase, was used to amplify tracer in order to 
improve the proportion of full-length sequences (see Note 4). 

1 . 5 u.L of synthesized cDNA was used as template and mixed with 1 u.L of Advantage 
cDN A polymerase mix, 5 uE of reaction buffer, 1 nmole of dNTP and 20 pmole of tracer 
primer (see Subheading 2.2.). Add water to a total volume of 50 uE. 

2. PCR condition: 95°C for 1 min; 95°C for 10 s, 68°C for 4 min, repeat for 15 cycles; 68°C 
for 10 min (see Note 5). 

3.3. Preparation of Driver 

3.3. 1. First-Strand cDNA Synthesis (see Note 6) 

The first-strand cDNA of driver was synthesized by using AMV reverse tran- 
scriptase according to the routine method (7) and the manufacturer's instruction 
manual. 

1. Elute about 1 (xg of mRNA in 10 uE of nuclease-free water and mix with 4 uE of 5X 
AMV RT reaction buffer, 1 uE of 10 mM dNTP, 2 uE of 40 mM sodium pyrophosphate, 
1 uE of 100 \iM oligo(dT), 2 uE of AMV RT. Mix well and incubate at 42°C for 1 h. 

2. Inactivate AMV RT by boiling the reaction for 3 min. 

3.3.2. Preparation of Driver Labeled with Biotin 

Single-strand driver was amplified by primering with single primer and labeled 
with biotin-21-dUTP simultaneously. In 100 p,L PCR reaction volume, add 40 nmole 
of dNTP, 2 nmole of Biotin-21-dUTP, 100 pmole of 3' anchoring primer (see Sub- 
heading 2.3.), 2 \iL of synthesized cDNA, 20 U of Taq polymerase, 10 [iL of 10X 
PCR buffer. PCR condition: 95°C for 10 s, 42°C for 2 min, 72 C for 1 min and repeat 
for 40 cycles. 



104 Huang, Yuan, and Cao 

3.4. Subtractive Hybridization 

3.4. 1. First Round of Subtractive Hybridization 

1 . The first-round hybridization was performed with 5 [xL of amplified tracer (see Subhead- 
ing 3.2.) and 100 uL of amplified driver (see Subheading 3.3.). Add 20X SSC to a final 
concentration of 3X SSC and add SDS to a final concentration of 0.1%. 

2. Incubate the reaction at 65°C for 12 h. 

3. Fill the spin column with 1 mL of Sephacryl S-400, wash Sephacryl S-400 with 1 mL of 
TEN by centrifugation at 1600g for 4 min. 

4. Repeat washing twice. 

5. Apply 50 uLof reaction to the top of the gel and centrifuge at 1600gfor4min. Collect the 
eluted sample. 

6. Wash 1 mL of S A-PMPs with equal volume of 0.5X SSC and suspend in 200 uL of 0.5X 
SSC. Mix the SA-PMPs with the sample obtained from step 5. Incubate the mixture at 
room temperature for 5 min and then incubate at 65°C for 5 min. 

7. Put the mixture in magnetic field instantly and collect the supernatant after the SA-PMPs 
congregated into pellet. This supernatant would be used as template in second-round 
hybridization. 

3.4.2. Second- and Third- round Subtractive Hybridization 

1. Amplify first round subtractive sample as follows: mix 5 |xL of template with 1 [xL of 
Advantage cDNA polymerase mix, 5 uL of reaction buffer, 10 nmole of dNTP and 20 pmole 
of PCR primer in 50 (xL reaction volume; run the PCR at 95 C C for 1 min, then 95°C for 
10 s, 68°C for 4 min, repeat for 15 cycles, followed with extension at 68°C for 10 min. 

2. Mix 5 [xL of the amplified reaction with 100 uL of amplified driver (see Subheading 3.3.), 
and perform the subtractive hybridization as described in Subheading 3.4.1. 

3. Repeat steps 1 and 2 once (see Note 7) 

4. The supernatant obtained from the final round subtractive hybridization would be used as 
the template for cloning differentially expressed genes. 

3.5. Cloning of Differentially Expressed Genes 

1. The subtracted template was amplified by PCR with AmpliTaq GOLD polymerase. Mix 
20 (xL of subtracted template with 10 nmole of dNTP, 20 pmole of tracer primer (see 
Subheading 2.2.), 2 U of AmpliTaq GOLD polymerase, 5 uL of GeneAmp 10X PCR 
buffer, 3 mM MgCl 2 , adjust the reaction volume to 50 |xL with water. PCR condition: 
94°C for 10 min; 95°C for 10 s and 68°C for 3 min, repeat for 20 cycles; 72°C extension 
for 10 min. 

2. Run the PCR products on 1.2 % agarose and retrieve the DNA ranging from 1 to 3 kb. 

3. Clone the retrieved DNA into pGEM-T vector and transform DH5a competent cells. 
Select transformants by blue/white selection and restriction enzymes digestion. 

4. Mix 100 ng of purified plasmid DNA with 1.6 pmole of sequencing primers and 4 uL of 
BigDye terminator ready reaction mix in 10 [xL of reaction volume. The sequencing PCR 
condition is 96°C for 10 s, 50°C for 5 s, 60°C for 4 min, repeat for 25 cycles. 

5. Add 1 [xL of 3 M NaAc, 25 fxL of 95% ethanol into the sequencing reaction. Mix them 
well and incubate at 4°C for 15 min. Centrifuge the mixture at 12,000g for 15 min at 4°C. 
Aspirate the supernatant carefully, add 150 (xL of 70% ethanol and centrifuge at 12,000g 
for 10 min at 4°C. Aspirate the supernatant and dry the pellet briefly. 

6. Dissolve the pellet in 2 uL of loading buffer, denature at 90°C for 2 min and load onto gel 
for sequencing. 



Full- Length Subtractive Cloning 105 

3.6. Evaluation of the Efficiency of Subtraction 

In our experiments, the efficiency of subtraction was evaluated by dot blotting with 
BluGene® nonradioactive nucleic acid detection system (Gibco-BRL) (see Note 8). 

1. Adjust the concentration of DNA samples to be detected to 100 ng/uL. Denature the 
samples by boiling for 2 min and spot 1 uL of each sample on the 1 -cm 2 squares of nylon 
membranes. 

2. Dry membranes for 2 h at 80 C C. 

3. Prepare biotin-labeled DNA probes by purifying driver with repeated ethanol precipitation. 

4. Soak the membrane in 2X SSC. 

5. Add freshly denatured sheared herring sperm DNA to prehybridization solution, mix, 
and place in a Hybritube (Gibco-BRL) with the membranes. Incubate at 42°C water 
bath for 2 h. 

6. Add heat-denatured probe and herring sperm DNA to hybridization solution at probe 
concentration of 100 ng/mL and mix. Remove prehybridization solution and add the 
hybridization solution. Hybridize at 42°C overnight. 

7. Wash the membranes with 250 mL of 2X SSC/0.1% SDS for 3 min at room temperature 
twice, with 250 mL of 0.2X SSC/0.1% SDS for 3 min at room temperature twice, and 
then with 250 mL of 0.16X SSC/0.1% SDS for 15 min at 65°C twice. Finally rinse the 
membranes in 2X SSC briefly. 

8. Rinse the membranes in Buffer 1. 

9. Incubate the membranes at 65°C for 1 h in Buffer 2. 

10. Dilute SA-AP conjugate to 1 jig/mL with Buffer 1 and incubate the membranes in this 
diluted SA-AP conjugate at room temperature for 10 min with gentle agitation. 

11. Wash the membranes with 250 mL of Buffer 1 for 15 min at room temperature, and with 
100 mL of Buffer 3 for 10 min at room temperature. 

12. Prepare the dye solution for each membrane by mixing 33 uL of NBT solution and 25 uL 
of BCIP solution in 7.5 mL of Buffer 3. Incubate the membranes in dye solution for 
30 min in dark. 

13. Wash the membranes with stopping buffer. 

4. Notes 

1. The method described here is based on our experiments of cloning differentially 
expressed genes from immunocytes, such as monocytes, dendritic cells (8,9), bone mar- 
row derived stromal cells, etc. Figure 1 shows the schematic diagram of the method. 
Several full-length genes have been cloned by this method and confirmed to be 
expressed differentially (10-12). 

2. Other equivalent purification systems can be used. The volume of GTC extraction buffer 
relies on cell types as well as cell numbers. In our experiments, because lymphocytes are 
of small size and contain less plasma, the 400 uL of GTC extraction buffer is sufficient 
for 1 x 10 7 PBMCs. 

3. It is noticeable that the efficiency of first strand cDNA synthesis is about 10-30% accord- 
ing to the reverse transcriptase to be used, which indicates that higher amounts of starting 
mRNA should be used if displaying of multiple genes or the genes expressed in low 
abundance is desired. 

4. All the PCRs in this experiment were performed on a Perkin-Elmer DNA thermal cycler 
model 9600. AmpliTaq GOLD polymerase also worked well in amplifying tracer in our 
experiment. 



106 Huang, Yuan, and Cao 

TRACER I Cap-finder pri^ |— — ~ 1 DRIVER 



SSS3 Q Q &- 



Cloning , Sequencing, Analysis 



Fig. 1. The schematic diagram of the strategy for cloning of cell differentially expressed 
genes with full-length directly. 



5. The extension time relies on not only the expected sequence size being cloned, but also 
the limitation of the techniques used in cloning and sequencing. The amplification cycles 
should be minimized in order to reduce PCR mutations. 

6. In the preparation of driver, the accuracy of DNA polymerase is less important than the 
efficiency of DNA amplification and biotin labeling, and short fragments are favorable in 
hybridization, so the requirements for reverse transcriptase and polymerase are different 
from those for tracer preparation. 

7. The template amount used in amplification and the PCR cycles should be adjusted 
according to the amount of products that obtained in subtractive hybridization, which 
should be monitored by electrophoresis in each step. Figure 2 shows the subtracted cDNA 
amplification of PBMCs stimulated with PHA. 

8. There are 60 positive clones among the 188 clones detected, so the proportion of specific 
clones is about 63.83%. Considering the frequency of Alu sequences (13), the efficiency 
of this subtractive cloning strategy is appropriate for cloning of differentially expressed 
genes. 

References 

1. Sagerstrom, C. G., Sun, B. I., and Sive, H. L. (1997) Subtractive cloning: past, present, 
and future. Annu. Rev. Biochem. 66, 751-783. 

2. Furuichi, Y. and Miura, K. (1975) A blocked structure at the 5' terminus of mRNA from 
cytoplasmic polyhedrosis virus. Nature 253, 374,375. 

3. Chenchik, A., Moqadam, F., and Siebert, P. (1996) A new method for full-length cDNA 
cloning by PCR, in A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis, (Krieg, 
P. A., ed.), Wiley-Liss, New York, pp. 273-321. 



Full-Length Subtractive Cloning 107 

i 2 3 4 5 6 




Fig. 2. The Subtracted cDNA Amplification of PBMCs Stimulated with PHA. 1 .2% agarose 
gel shows amplified products of three rounds of subtractive hybridization. Lane 1: 1 kb DNA 
ladder (Gibco, cat. no. 15615); Lane 2: PCR products by using 0.02 uL reverse-transcribed 
products as a template; Lane 3, 4, 5: the first, second and third subtractive PCR products by 
using 40 |iL subtracted cDN A as template; Lane 6: 1 00 bp DNA ladder (Gibco, cat. no. 15628). 



4. Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high 
yield from ( bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216-2220. 

5. Thompson, J., Solomon, R„ Pellegrino, M., Sakai, K., Lewin, M., Field, M., et al. (1989) 
A noise-free molecular hybridization procedure for measuring RNA in cell lysates. Analyt. 
Biochem. 181,371-378. 

6. Swaroop, A., Xu, J. Z., Agarwal, N., and Weissman, S. M. (1991) A simple and efficient 
cDNA library subtraction procedure: isolation of human retina-specific cDNA clones. 
Nuel. Acids Res. 19, 1954. 

7. Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Reverse transcriptase (RNA-dependent 
DNA polymerase), in Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring 
Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 5.52-5.55. 

8. Zhao, Z., Huang, X., Li, N., Zhu, X., and Cao, X. (1999) Screening and cloning of new 
genes differentially expressed in dendritic cells in large scale. Chin. J . Biochem. Mol. 
Biol. 15, 624-628. 

9. Zhao, Z., Huang, X., Li, N., Zhu, X., Chen, S., and Cao, X. (1999) Direct cloning of cell 
differential expression genes with full-length by a new strategy based on the multiple 
rounds of "long distance" polymerase chain reaction and magnetic beads mediated sub- 
traction. J. Biotech. 73, 35-41. 

10. Huang, X., Yuan, Z., Chen, G., Zhang, M., Zhang, W., Yu, Y., and Cao, X. (2001) Cloning 
and characterization of a novel ITIM containing Lectin-like immunoreceptor LLIR and its 
two transmembrane region deletion variants. Biochem. Biophys. Res. Commun. 281, 131-140. 

11. Huang, X., Zhao, Z., Yuan, Z., Zhang, M., Zhu, X., Chen, G., and Cao, X. (2000) Cloning 
and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleopro- 
tein M4 from human dendritic cells. Science In China (Series C) 43, 648-654. 



108 Huang, Yuan, and Cao 

12. Li, N., Huang, X., Zhao, Z., Chen, G., Zhang, W., and Cao, X. (2000) Identification and 
characterization of a novel gene KE04 differentially expressed by activated human 
dendritic cells. Biochem. Biophys. Res. Commun, 279, 487-493. 

13. Claverie, J. M. and Makalowski, W. (1994) Alu alert. Nature 371, 752. 



II 

Cloning PCR Products 



12 

Cloning PCR Products 

An Overview 

Baotai Guo and Yuping Bi 

1. Introduction 

A successful cloning of polymerase chain reaction (PCR)-derived DNA fragment 
is a key step for further analysis of the amplified DNAs, though it is often a difficult 
task. Many cloning methods have been established; various commercial cloning kits 
are also available. These methods can be separated into two groups: ligation-depen- 
dent cloning and ligation-independent cloning. The former is used more widely than 
the latter. According to DNA ends, the existing ligation-dependent cloning methods 
for PCR products can be further divided into three types: blunt-end cloning, sticky- 
end cloning, and T-A cloning. 

To choose a suitable ligation-dependent cloning method, several factors should be 
taken into account. First, consider the purpose of your cloning manipulation. For exam- 
ple, if the selected clones are to be expressed into proteins, then the directional cloning 
in an expression vector is desirable. Second, choose or prepare a suitable vector that 
matches with your unmodified or modified PCR products. Different DNA polymerases 
generate PCR products with different DNA ends, which are compatible to blunt-end 
or sticky-end cloning vectors. Third, for cloning extremely long PCR products (e.g. 
more than 10 kb), which are beyond the cloning capacity of the commonly used plas- 
mid vectors, utilize cosmid or X.DNA vectors. To facilitate the selection of the clones 
with the inserts and improve the cloning efficiency, an efficient screening system for 
the positive clones is also very useful. 

In this chapter, various cloning methods will be briefly reviewed; their advantages 
and disadvantages are pointed out. The strategies for positive selection are also dis- 
cussed briefly. 



From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 



111 



1 12 Guo and Bi 

2. Ligation-Dependent Cloning 
2. 1. Blunt-End Cloning 

Blunt-end cloning is a conventional technique used in cloning PCR-amplified DNA 
fragments as well as any other blunt-end and double-stranded DNAs. In this method, 
PCR products with blunt ends and the blunt-cut (often dephosphorylated) vectors are 
ligated together. DNA fragments are inserted into the vectors in both orientations, 
therefore, directional cloning is not available. 

Thermostable DNA polymerases, such as Vent, possess 3'-5' exonuclease activity, 
the so-called proofreading activity, which generates blunt-end PCR products (1). The 
resultant DNAs are relatively easy to be directly cloned through blunt-end ligation, 
because they are compatible with the blunt-end vectors. On the other hand, DNA poly- 
merase like Taq exhibits nontemplate terminal transferase activity, which can add one 
deoxynucleotide to the 3'-hydroxyl termini of double-stranded PCR products and pro- 
duce DNA fragments with single 3 '-overhang nucleotide (2). The presence of 3 '-over- 
hangs prohibits the ligation of these fragments with blunt-end plasmid vectors. 
Therefore blunt-end cloning strategy is problematic with PCR products from DNA 
polymerases such as Taq and Tth. To overcome the cloning difficulty, PCR products 
may be treated with Klenow fragment of DNA polymerase I (3), T4 (4,5) or Pfu (4) 
polymerase to polish the ragged DNA ends, which can greatly increase the ligation 
and cloning efficiency. 

Generally speaking, blunt-end cloning of PCR products is not as efficient as sticky- 
end cloning because of the inefficiency of blunt-end ligation and high tendency of 
self-ligation of vectors, which yields lower recombinant clones. Besides removal of 
3'-overhangs, purification of PCR products, and addition of excessive T4 DNA ligase 
could also improve the blunt-end ligation efficiency. Adding restriction enzymes with 
a rare recognition sequence to ligation mixture can prevent the linearized vectors from 
recirculation during ligation reaction, resulting in lower self-ligation of vectors and 
higher recombinant clones (6). To facilitate the blunt-end cloning of PCR products, a 
blunt-end cloning procedure was established for cloning PCR products up to 10 kb (7). 
The cloning steps include an optimized polishing (if necessary) / kinase treatment of 
amplified DNAs, and ligation between the polished DNAs and the blunt dephosphory- 
lated plasmid vectors (to inhibit self-ligation). PCR products from any DNA poly- 
merase can be cloned using this method, and the cloning efficiency is very high. In 
addition, introduction of a positive selection system could further increase the cloning 
efficiency. Because only recombinants containing inserted DNA can survive the selec- 
tion, the self-ligation of vectors is no longer a problem. 

Blunt-end cloning takes advantages of ease in manipulation, low expenses, and 
lack of addition of a base to the amplified sequence owing to the 3'-overhangs pro- 
duced by polymerase like Taq, which is especially useful in site-directed mutagenesis 
analysis (4). Because there is no need to incorporate restriction recognition sequences 
or other special sequences to the PCR primers, many researchers still think blunt-end 
cloning is an attractive cloning approach and prefer to use it. 



Cloning PCR Products 1 13 

2.2. Sticky-End Cloning 

Sticky-end cloning is routinely used to clone PCR products in many laboratories. 
Restriction sites can be introduced into the 5' end of the PCR primers. As amplifica- 
tion reaction proceeds, the primers are integrated into the PCR products. After diges- 
tion by the corresponding restriction enzymes, sticky ends form at both ends of the 
PCR products. Digested PCR products and equivalently cut vectors can be ligated 
with very high efficiency, resulting in fairly efficient sticky-end cloning. Restriction 
sites introduced into the two PCR primers can be identical or different. If they are 
different, then the PCR products may be inserted into the vectors in only one orienta- 
tion, enabling directional cloning of these products (8,9). 

Some problems are frequently encountered with sticky-end cloning. Many restriction 
endonucleases fail to efficiently cleave the restriction recognition sites located at the 
5' ends of double-stranded PCR products, especially when the sites are near or extremely 
near the 5' ends (10,11), which may be a result of the unstable binding of the restriction 
enzymes to the DNA termini. One solution is to add extra nucleotides to the 5' end 
outside the restriction site, but this increases the cost of primer synthesis and may also 
decrease the binding efficiency of the primers to the templates. For sticky-end clon- 
ing, another problem is the undesirable cleavage of internal restriction site(s) identical 
to those in primers. This problem is hard to predict, because the insert sequences are 
often unknown. The third problem is the generation of primer dimers. Restriction rec- 
ognition sequences are commonly inverse repeat sequences (palindromes) that facili- 
tate formation of primer dimers, which decrease amplification efficiency. 

Another solution to overcome the restriction cleavage difficulty is by PCR product 
concatenation. The primary PCR products generated with 5'-phosphorylated primers 
can be first self-ligated by the T4 DNA ligase to form DNA concatamers, then 
the concatamerized DNAs can be readily cleaved, resulting in a higher efficiency 
sticky-end cloning (11). In another approach, only half of the palindromic restriction 
site is incorporated into one primer, and the other half into another primer. After con- 
catamerization of the PCR-derived DNAs, the entire site is reconstituted, resulting in 
an easy sticky-end cloning as aforementioned (11). In both methods, the key is to 
convert the terminal sites into internal sites, nevertheless, directional cloning is not 
possible and both are intolerant to 3'-overhangs in PCR products. In addition, more 
steps are required after PCR, so the manipulation is laborious. 

In 1999, an autosticky PCR (AS-PCR) cloning method was reported (9). This 
method uses two special primers that contain tetrathydrofuran derivative abasic sites, 
which makes DNA polymerase stall. The PCR products are therefore directly of dif- 
ferent single-stranded DNA (5'-overhang) at both ends, corresponding to sticky ends 
of two different restriction sites. The intact PCR products can be directionally cloned 
into correspondingly cut plasmid vectors. While retaining the advantages of common 
sticky-end cloning, this method needs no post-PCR processing steps, avoiding the 
sensitivity of the internal sites to restriction enzymes and cleavage difficulty of sites 
at DNA ends. 



1 14 Guo and Bi 

Sticky-end cloning is really a personalized manipulation, and many flexible designs 
are employed to meet various cloning demands in different laboratories. 

2.3. T-A Cloning 

Taq is the most widely used thermostable DNA polymerase, and it has terminal 
transferase activity. In the presence of four dNTPs, deoxyadenosine (dA ) is preferen- 
tially added to the 3' termini of PCR-amplified duplex DNAs, leaving a single 3' dA 
overhang (2). Without modification by restriction enzymes or any other enzymes, PCR 
products with 3' dA overhangs can be directly cloned into a linearized plasmid vector 
with complementary single 5' deoxythymidine (dT) overhangs at both ends. The dT 
overhang-containing vector is called T-vector, and the corresponding cloning is named 
T-A cloning. In fact, T-A cloning is also a sticky-end cloning, but a special one with 
only one base in the sticky end. 

T-vectors can be prepared in laboratories through enzymatic reactions. By taking 
advantage of the terminal transferase activity of Taq polymerase, when only 
deoxythymidine triphosphate (dTTP) is present in the reaction, dT can be uniquely 
added to the 3' termini of a blunt-ended plasmid, resulting in a T-vector (12). A similar 
method is used to make T-vector in the presence of ddTTP, in which DNA terminal 
transferase introduces only one ddTTP to 3' termini of linear plasmids that are opened 
up with blunt-cutting restriction enzymes (13). The yielding T-vector does not possess 
3'-hydroxyl group, so phosphodiester bond cannot form between 3' end of vector and 
5' end of DNA fragment. The recombinant plasmid is of two nicks, but there is not 
much impact on transformation. 

T-vector can also be produced directly by cutting plasmids with selected enzymes. 
Restriction enzymes, such as Xcml, Hphl, and Ahdl , generate single 3' dT overhang in 
the digested DNAs. Xcml recognition sequences are not present in the multiple clon- 
ing sites of commonly used plasmids, so it is convenient to add the Xcml sites to the 
desired plasmids in order to create the T-vector. The recognition sequence of Xcml is 
5' CCANNNNNNNNNTGG 3', the internal bases (N) are alterable. Two adjacentXcml 
sites with appropriate sequences are needed for the T-vector creation (14-18). T-vec- 
tor is formed simply by Xcml digestion. An example of T-vector preparation by Xcml 
digestion is shown in Fig. 1. 

T-A cloning is simple, reliable, and more efficient than blunt-end cloning. As long 
as DNA polymerases without 3-5' exonuclease activity are used, T-A cloning is 
applicable and the PCR products may need no post-PCR processing steps. Therefore, 
T-A cloning is the most widely used cloning method for PCR products. It can be also 
used in cloning DNA fragments from other PCR-based techniques, such as RAPD 
(random amplified polymorphic DNA) and AFLP (amplified fragment length poly- 
morphism). It has been used in establishing specific SCAR (sequence-characterized 
amplified region) markers (19,20) for molecular identification or recovery of DNA 
bands of interest in AFLP analyses. T-A cloning proves to be convenient and reliable 
in these applications. Although DNA polymerase with proofreading activity does not 
produce 3'-overhangs, their PCR products can still be cloned into T-vector after con- 
ducting a tailing procedure using Taq polymerase prior to ligation, which makes T-A 
cloning a universal method applicable to all DNA polymerases. 



Cloning PCR Products 1 15 



5'NNNCCAAGCTTCCCATGGNNNNNNCCATGTCATGAGTGGNNN3' 
JGTTCGAAGGGTACCN NNNN NGGTACAG 
Xcm I Xcm I 



the modified 

a'NNNGGTTCGAAGGGTACCNNNNNNGGTACAGTACTCACCNNNS' P lasmid 



1 



Xcm I digestion 



T-vector 



5'NNNCCAAGCTT TGAGTGGNNN3' 

3'NNNGGTTCGA TACTCACCNNN5' 

+ 
5'CCCATGGNNNNNNCCATGTCA3' ^^ 

3'AGGGTACCNNNNNNGGTACTG5' ^9™** 

Fig. 1 . Preparation of T-vector by Xcml digestion 

Although it is not required to do any post-PCR processing in T-A cloning, some- 
times it is helpful to purify the PCR products before ligation to remove primer dimers, 
which compete favorably with the PCR products for the T-vector. In addition to the 
disadvantages of nondirectional cloning and the need for T-vector, T-A cloning also 
has the drawback of variable cloning efficiencies. This presumably is a consequence 
of variability in 3'-dA addition to PCR products mediated by Taq polymerase. It was 
found that not only dA but also dG, dC, or dT can be added to the 3' ends of PCR 
products (21), and the dA addition frequency is determined by the 3' end base and the 
adjacent bases in DNA fragments (22). It also appears that the dT overhangs in the 
T-vector and dA overhangs in the PCR products are unstable and may get lost during 
storage, especially at room temperature. The typical characteristics of blunt-end, 
sticky-end, and T-A cloning methods are summarized in Table 1. 

3. Ligation-lndependent Cloning 

Conventional cloning means DNA recombination in vitro, that is, creation of 
phosphodiester bonds between DNA fragments and vectors by DNA ligase in vitro. 
Ligation-independent cloning (LIC), on the other hand, does not involve in vitro 
ligation, so it is ligase-free cloning. Various LIC methods exist, and different cloning 
strategies are adopted. PCR products and linearized vector containing identical 
terminal sequences to them can be cotransferred into Escherichia coli strain JC8679, 
then DNA recombination occurs in vivo by the help of the host homologous recombi- 
nation system (23). This in vivo cloning (IVC) method is simple, but the host strains 
are very restricted. Like conventional sticky-end cloning, some LIC methods require 
the creation of sticky ends in both the inserts and the vectors. But the differences are 
that the sticky ends in the PCR amplification products are not created by the restriction 
enzymes, but by various other DNA enzymes, and the extra sequences at the 5' end of 
the primers are usually longer than common restriction recognition sequences. This 
type of cloning is called enzymatic modification-mediated LIC. In another LIC called 
PCR-induced LIC, the PCR products and the plasmids are fused together in a second 
PCR reaction, independent of any enzymatic modifications. In LIC the resulting 



116 



Guo and Bi 



Table 1 

Comparison of Three Ligation-Dependent Cloning Methods for PCR Products 



Method 



Vector Primers PCR Products 3'-overhang Ligation Advantages 



Blunt-End Common Common Intact Intolerant 

Sticky-End Common Additional Additional Tolerant 
Sequences Sequences 



T-A 



T-vector Common Additional A Tolerant 



Less Simple and 

efficient Universal 

Efficient Directional 
Cloning 

Efficient Simple and 

High Efficiency 



recombinant DNAs are open circular DNA molecules with nicks, but they can be 
directly applied to transform bacterial competent cells with high efficiency. 

3. 1. Enzymatic Modification-Mediated LIC 

In enzymatic modification-mediated cloning, special sequences are introduced into 
5' end of PCR primers. The PCR products are then treated with various DNA enzymes, 
such as T4 DNA polymerase (24), exonuclease III (25), or uracil DNA glycosylase 
(UDG) (26-28) to generate single-stranded tails (sticky ends), which will hybridize 
with the complementary sticky ends in vectors, forming a relatively stable recombi- 
nant plasmid. When using T4 DNA polymerase, additional 12 nucleotide sequences 
lacking dCMP are incorporated into the 5' end of primers, the resultant PCR products, 
lacking dGMP at their 3' ends, are treated by T4 DNA polymerase to form 5' over- 
hangs in the controlled reaction conditions because of its 3'-5' exonuclease activity 
(24). Exonuclease III is also efficient to create 5'-overhangs in PCR products (25). 
With UDG-mediated LIC (27), 12-base dUMP-containing sequences are added to the 
5' end of the PCR primers, resulting in the selective replacement of dUMP residues 
into 5' end of PCR products. After UDG treatment, which selectively degrades dU in 
the PCR products, 3'-overhangs are generated. Unlike many restriction enzymes, UDG 
functions effectively near DNA termini, thus enabling high efficiency degradation and 
subsequent cloning. Directional cloning can be accomplished by adding different extra 
sequences to the two PCR primers (25,27). These methods are highly efficient, but 
require long additional sequences in primers and post-PCR enzymatic treatments. 

3.2. PCR-lnduced LIC 

By the skillful use of PCR techniques, it is possible to join any DNA fragments and 
vectors together. In PCR-induced LIC, cloning of PCR products is mediated by further 
PCR, in contrast to the conventional ligation mediated by DNA ligase. One strategy is 
that inserts and vectors are amplified separately and the two amplification products 
are then mixed, denatured, and annealed to form a recombinant DNA (29). Another 
strategy includes four pairs of primers, but inserts are first amplified with primers of 
additional sequences at the 5' ends that result in PCR products whose 3' ends are 
complementary to the 3' ends of the recipient linearized plasmid, then mixed with this 
linearized plasmid, followed by second PCR to produce nicked cyclic recombinant 



Cloning PCR Products 1 1 7 

DNAs (30,31). These methods are efficient and fast, but more than one primer pair is 
needed and two PCR amplifications are conducted. They are very useful in studies of 
site-directed mutagenesis and in vitro recombination (29), but researchers should be 
aware of the complexity of the design of additional sequences at the 5' end of the 
primers and the matching of these sequences with the corresponding vector sequences. 

Still based on PCR-induced strategy, Chen et al. developed a new cloning method 
that enables inserting a PCR product into a vector flexibly and precisely at any desired 
location with high efficiency (32). For details, see the corresponding chapter in this 
section. 

In LIC methods, PCR primers contain extra sequences for combination of PCR 
products with vectors or sticky-end creation, and the primers are usually longer than 
those used in conventional sticky-end cloning. Because of the need for special 
sequences in primers, LIC methods are hardly useful in cloning fragments from RAPD 
and AFLP. 

4. Positive Selection System 

As in general cloning, the cloning efficiency of PCR products is also dependent on 
three factors: ligation efficiency, transformation efficiency of competent cells, and 
selection efficiency of desired transformants. In most cases, transformation efficiency 
is not a limiting factor, but ligation and selection of positive transformants are. 
Improving the selection efficiency will improve the entire cloning efficiency. Based 
on the a-complementation of (3-galactosidase, the blue/white screening system is 
widely used in DNA cloning, including PCR products cloning. This method allows 
visual color discrimination of recombinants from nonrecombinants in the presence of 
X-gal and IPTG in bacterial culture medium. One disadvantage is that it can lead to a 
very high background of nonrecombinant clones because all self-ligated vectors can 
be transformed into host and produce blue colonies. Another disadvantage is that 
in-frame cloning also leads to false-negative blue colonies. 

To decrease the background and facilitate the selection of recombinants, many 
positive screening vectors or systems have been established (33-36). They use the 
strategy of insertional inactivity of lethal gene such as ccd B, which encodes potent 
poison protein to gyrase (33), and bam, which encodes barnase (35), or toxic sensi- 
tivity gene (36). Positive system creates powerful selection, because the bacteria 
with self-ligated vectors carrying lethal or toxin sensitive gene fail to form colonies 
on the plate. Because of the limitations in host range, requirements for special com- 
ponents in medium or limited cloning sites, positive selection systems are not used 
widely in PCR products cloning. 

In a new positive-selection method applicable to clone PCR products up to 9 kb 
(37), the plasmid vector harbors the lethal mutant gene crp s encoding an altered catabo- 
lite gene activator protein CAP S . CAP is also known as cyclic AMP receptor protein 
CRP. CAP s -cAMP complex is toxic to host cells, so wild-type cells with crp s are killed. 
Only recombinant plasmids containing inserts in the unique restriction site within crp s 
gene to make it disrupted can survive, leading to a very strict selection for recombi- 
nant transformants. The advantages of this method are of no need for special compo- 
nents in culture medium and suitable for a broad range of host bacterial strains, but the 



1 18 Guo and Bi 

vector must be maintained in E. coli cycr strains, which are unable to produce adeny- 
late cyclase. A commercial kit from Roche Biochemicals is also based on mutant crp 
gene, which allows efficient blunt-end cloning of PCR products. 

Another positive screening system was specially developed to clone PCR products 
(38). In this method, the vector with a translation deficiency in lacZo. gene is created 
by deletion of the Shine-Dalgarno sequence and initiation codon. Instead, the Shine- 
Dalgarno sequence and initiation codon are incorporated into one of the PCR primers 
to allow complementation of inactive lacZa gene by the PCR products, which result in 
blue transformed bacterial colonies. 

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1 . Lohff, C. J. and Cease, K. B. (1992) PCR using a thermostable polymerase with 3'to 5' exonu- 
clease activity generates blunt products suitable for direct cloning. Nucl. Acids Res. 20, 144. 

2. Clark, J. M. (1988) Novel non-templated nucleotide addition reactions catalyzed by pro- 
caryotic and eucaryotic DNA polymerases. Nucl. Acids Res. 16, 9677-9686. 

3. Hemsley, A., Arnheim, N., Toney, M. D., Cortopassi, G., and Galas, D. J. (1989) A simple 
method for site directed mutagenesis using the polymerase chain reaction. Nucl. Acids 
Res. 17,6545-6551. 

4. Costa, G. L. and Weiner, M. P. (1994) Polishing with T4 or Pfu polymerase increases the 
efficiency of cloning PCR fragments. Nucl. Acids Res. 22, 2423. 

5. Skryabin, B. and Vassilacopoulou, D. (1993) A simple and fast method for cloning and 
analyzing polymerase chain reaction products. Genet. Analyt. Tech. Appl. 10, 1 13-115. 

6. Costa, G. L., Grafsky, A., and Weiner, M. P. (1994) Cloning and analysis of PCR-gener- 
ated DNA fragments. PCR Meth. Appl. 3, 338-345. 

7. Novy, R. E., Yaeger, K. Y., and Kolb, K. (1996) Perfectly blunt cloning: a superior method 
for cloning PCR products or any DNA. Innovations 6, 71 1. 

8. Scharf, S. J., Horn, G. T., and Erlich, H. A. (1986) Direct cloning and sequence analysis of 
enzymatically amplified genomic sequences. Science 233, 1076-1078. 

9. Gal, J., Schnell, R., Szekeres, S., and Kalman, M. (1999) Directional cloning of native 
PCR products with preformed sticky ends (autosticky PCR). Mol. Gen. Genet. 260, 569-573. 

10. Kaufman, D. L. and Evans, G. A. (1990) Restriction endonuclease cleavage at the termini 
of PCR products. BioTechniques 9, 304-306. 

11. Jung, V., Pestka, S. B., and Pestka, S. (1990) Efficient cloning of PCR generated DNA 
containing terminal restriction endonuclease recognition sites. Nucl. Acids Res. 18, 6156. 

12. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Construction of T-vec- 
tors, a rapid and general system for direct cloning of unmodified PCR products. Nucl. 
Acids Res. 19, 1154. 

13. Holton, T. A. and Graham, M. W. (1991) A simple and efficient method for direct cloning 
of PCR products using ddT-tailed vectors. Nucl. Acids Res. 19, 1 156. 

14. Mead, D. A., Pey, N. K., Herrnstadt, C, Marcil, R. A., and Smith, L. M. (1991) A universal 
method for the direct cloning of PCR amplified nucleic acid. Biotechnology 9, 657-663. 

15. Cha, J., Bishai, W., and Chandrasegaran, S. (1993) New vectors for direct cloning of PCR 
products. Gene 136, 369,370. 

16. Harrison, J., Molloy, P. L., and Clark, S. J. (1994) Direct cloning of polymerase chain 
reaction products in an Xcm I T- vector. Analyt. Biochem. 216, 235-236. 

17. Kwak, J. H. and Kim, M. Y. (1995) Construction of T-vector for direct cloning and expres- 
sion of cloned genes in Escherichia coli. Analyt. Biochem. 228, 178-180. 

18. Borovkov, A. Y. and Rivkin, M. I. (1997) Xcm I-containing vector for direct cloning of 
PCR products. BioTechniques 22, 812,813. 



Cloning PCR Products 1 19 

19. Hermosa, M. R., Grondona, I., Diaz-Mingues, J. M., Iturriaga, E. A., and Monte, E. (2001) 
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11, a biological control agent against soilborne fungal plant pathogens. Curr. Genet. 38, 
343-350. 

20. Xu, M., Huaracha, E., and Korban, S. S. (2001) Development of sequence-characterized 
amplified regions (SCARs) from amplified fragment length polymorphism (AFLP) mark- 
ers tightly linked to the Vf gene in apple. Genome 44, 63-70. 

21. Hu, G. (1993) DNA polymerase-catalyzed addition of nontemplated extra nucleotides to 
the 3' end of a DNA fragment. DNA Cell Biol. 12, 763-770. 

22. Brownstein, J. M., Carpten, J. D., and Smith, J. R. (1996) Modulation of non-templated 
nucleotide addition by Taq DNA polymerase: primer modifications that facilitate 
genotyping. BioTechniques 20, 1004-1006. 

23. Oliner, J. D., Kinzler, K. W., and Vogelstein, B. (1993) In vivo cloning of PCR products in 
E. coli. Nucl. Acids Res. 21, 5192-5197. 

24. Aslanidis, C. and de Jong, P. J. (1990) Ligation-independent cloning of PCR products 
(LIC-PCR). Nucl. Acids Res. 18, 6069-6074. 

25. Hsiao, K.C. (1993) Exonuclease III induced ligase-free directional subcloning of PCR 
products. Nucl. Acids Res. 21, 5528-5529. 

26. Nisson, P. C, Rashtchian, A., and Watkian, P. C. (1991) Rapid and efficient cloning of 
Alu-PCR products using uracil DNA glycosylase. PCR Meth. Appl. 1, 120-123. 

27. Rashtchian, A., Buchman, G. W., Schuster, D. M., and Berninger, M. S. (1992) Uracil 
DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: appli- 
cation to genomic and cDNA cloning. Analyt. Biochem. 206, 91-97. 

28. Smith, C, Day, P. J., and Walker, M. R. (1993) Generation of cohesive ends of PCR 
products by UDG-mediated excision of dU, and application for cloning into restriction 
digest-linearized vectors. PCR Meth. Appl. 2, 328-332. 

29. Jones, D. H., Sakamoto, K., Vorce, R. L., and Howard, B. H. (1990) DNA mutagenesis 
and recombination. Nature 344, 793,794. 

30. Shuldiner, A. R., Scott, L. A., and Roth, J. (1990) PCR-induced (ligase-free) subcloning: 
a rapid reliable method to subclone polymerase chain reaction (PCR) products. Nucl. Acids 
Res. 18, 1920. 

31. Shuldiner, A. R., Tanner, K., Scott, L. A., Moore, C. A., and Roth, J. (1991) Ligase-free 
subcloning: a versatile method to subclone polymerase chain reaction (PCR) products in a 
single day. Analyt. Biochem. 194, 9-15. 

32. Chen, G. J., Qiu, N., Karrer, C, Caspers, P., and Page, M. G. (2000) Restriction site-free 
insertion of PCR products directionally into vectors. BioTechniques 28, 498-500, 504,505. 

33. Bernhard, P., Gabant, P., Bahassi, E. M., and Couturier, M. (1994) Positive-selection vec- 
tors using the F plasmid ccd B killer gene. Gene 148, 71-74. 

34. Henrich, B. and Schmidtberger, B. (1995) Positive-selection vector with enhanced lytic 
potential based on a variant of (XI 74 phage gene. E. Gene 154, 51-54. 

35. Yazynin, S. A., Deyev, S. M., Jucovic, M., and Hartley, R. W. (1996) A plasmid vector 
with positive selection and directional cloning based on a conditionally lethal gene. Gene 
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36. Kast, P. (1994) pKSS-A second-generation general purpose cloning vector for efficient 
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37. Schlieper, D., von Wilcken-Bergmann, B., Schmidt, M., Sobek, H., and Muller-Hill, B. 
(1998) A positive selection vector for cloning of long polymerase chain reaction fragments 
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Res. 24, 3474,3475. 



13 

Using T4 DNA Polymerase 

to Generate Clonable PCR Products 

Kai Wang 

1. Introduction 

Polymerase chain reaction (PCR) mediated through Taq DNA polymerase has 
become a simple and routine method for cloning, sequencing, and analyzing genetic 
information from very small amount of materials (7). Taq DNA polymerase, like 
some other DNA polymerases, lacks 3' to 5' exonuclease activity and will add 
nontemplate-directed nucleotides to the ends of double-stranded DNA fragments 
(2). Because of the strong preference of the Taq polymerase for deoxyadenosine 
s'triphosphate (dATP), the nucleotide added is almost exclusively an adenosine. This 
results in generating "ragged" unclonable amplification products (3). Restriction 
endonuclease sites are often incorporated into the amplification primers so that 
clonable PCR products can be generated by restriction enzyme cleavage (4). How- 
ever, the possible secondary sites located within the amplified products often com- 
plicate the cloning and interpretation of PCR results. A cloning system exploiting 
the template-independent terminal transferase activity of Taq polymerase has been 
developed (5-7). However, a special vector with thymidine (T) overhanging ends 
has to be used in the process. 

T4 DNA polymerase has strong exonuclease and polymerase activities in a broad 
range of reaction conditions (8). By adapting its strong enzymatic activities, a simple 
and efficient method to generate clonable PCR fragments with T4 DNA polymerase 
has been developed. The T4 DNA polymerase not only repairs the ends of the PCR 
products, but also removes the remaining primers in the reaction with its strong single- 
stranded exonuclease activity. Therefore, this method does not require multiple sample 
handling, buffer changes, or gel purification steps. Instead, a simple alcohol precipita- 
tion step is used to purify the PCR products. 



From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 



121 



122 Wang 

2. Materials 

2.1. PCR 

1. DNA template containing the sequence interested. 

2. Oligonucleotide primers. 

3. Taq DNA polymerase. 

4. 10X PCR and enzymatic repair buffer: 500 mM Tris-HCl, pH 9.0, 25 inM MgCl 2 , 500 mM 
NaCl, and 5 mM dithiothreitol (DTT). Commercial 10X PCR buffer also works well. 

5. 1.5 mM 10X deoxynucleotides (dNTP) solution. Concentrated stock solution (100 mM) 
can be obtained from Pharmacia (Piscataway, NJ) or Boehringer Mannheim (Indianapo- 
lis, IN). 

6. Gel electrophoresis and PCR equipment. 

2.2. End Repair and Cloning 

1. Enzymes: T4 DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase. Enzymes 
can be purchased from Boehringer Mannheim, Life Technologies (Gaithersburg, MD) or 
any other provider. 

2. 1 mM of ATP solution. Concentrated stock solution (100 mM) can be obtained from 
Boehringer Mannheim. 

3. Isopropyl alcohol. 

4. Vector (blunt end and dephosphorylated). 

5. 10X Ligase buffer: 660 mM Tris-HCl, pH 7.6, 66 mM MgCl 2 , 10 mM adenosine (ATP), 
1 mM Spermidine, and 10 mM DTT. Commercially available 10X ligase buffer also 
works well. 

6. TE buffer: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA. 

7. 5MNaCl. 

3. Methods 

3. 1. Primer Design 

No special primer is needed, however, secondary structure and stretch of homopoly- 
mer should be avoided. 

3.2. PCR Reaction 

1. Prepare the following in a PCR reaction tube: 

a. 5 uL of 10X PCR buffer (500 mM Tris-HCl, pH 9.0, 25 mM MgCl 2 , 500 mM NaCl, 
and 5 mM DTT). 

b. 0.25 jig of DNA template such as genomic DNA. 

c. 1 (xM of each primer. 

d. 0.15 mM of each deoxynucleotides (dNTP). 

e. 1 U Taq polymerase (Perkin-Elmer, Cetus, Norwalk, CT). 

f. add deionized H 2 to a final volume of 50 uL. 

2. Amplification conditions largely depend on the specific applications. However, a general 
cycling profile listed below can be used in most of the amplifications. 

a. Initial denaturation 94°C for 5 min. 

b. Amplification (30 cycles) 94°C for 30 s; 55°C for 45 s; and 72°C for 90 s. 

c. Final extension 72 C C for 10 min. 

3. Examine the PCR amplification results with agarose gel electrophoresis (see Note 1). 



Clonable PCR Product Generation 123 

3.3. End Repair for Cloning 

1. Add the following to PCR reaction tubes directly to repair the PCR products (see Note 2): 

a. 1 U of T4 DNA polymerase; 

b. 1 \iL of 4 mM dNTP solution (optional) (see Note 3); 

c. 5 U of T4 polynucleotide kinase (see Note 4); 

d. 1 uE of 1 mM ATP. 

2. Incubate the reaction tubes at 25 C C (room temperature) for 20 min (see Notes 5 and 6). 
Stop the reactions by adding 3 uE of 0.5 M EDTA (pH 8.0). 

3. Incubate the reaction tubes at 70 C C for 10 min to inactive the enzymes. 

4. Precipitate the PCR products at room temperature by adding 5 uE of 5 M NaCl and 60 uE 
of isopropyl alcohol (3) (see Note 7). 

5. Resuspend the DNA fragments in 20 u.L of TE or water. 

6. Take 2 uE (containing approx 20-50 ng of PCR product) and mix with ligase and vector 
for ligation (see Notes 4 and 8): 1 uE of 10X ligase buffer; 1 uE of T4 DNA ligase; 2 uE 
of repaired PCR product; dephosphorylated vector DNA (60-150 ng); and add deionized 
H 2 to a final volume of 10 uE. 

7. Incubate at 16°C overnight. 

8. Dilute the ligation reaction fivefold In TE buffer. Use 2 uE of the diluted ligation reaction 
for transformation. 

4. Notes 

1 . In case of multiple PCR products from a single reaction was observed, the specific prod- 
ucts should be purified by gel electrophoresis based on its estimated size after repair 
reaction. Several different methods can be used to purify DNA fragments from agarose 
gel, such as phenol extraction from low-melting gel, "glassmilk" method, or by simple 
low-speed centrifugation. 

2. This protocol utilizes a single buffer for all the enzymes that include Taq polymerase in 
PCR, T4 polymerase, and T4 polynucleotide kinase in end repairing. Therefore, slightly 
higher concentration of reagents and enzymes can be added in the reaction. 

3. T4 DNA polymerase can be added directly into PCR tube without providing additional 
nucleotides. However, T4 DNA polymerase balances its exonuclease and polymerase 
activities based on the concentration of available deoxynucleotides. Depending on the 
length of amplification products, number of cycles, and nucleotide sequence composition 
of amplified region, the remaining nucleotide concentration after PCR amplification may 
be different from experiment to experiment. In order to avoid unnecessary confusion, 
supplemental nucleotides are routinely added for end-repair reaction. 

4. T4 polynucleotide kinase is not needed when vector used has not been treated with phos- 
phatase previously. However, dephosphorylated vector should be used to increase the 
cloning efficiency. 

5. Room temperature (25°C) was chosen for the reaction, because T4 DNA polymerase has 
excessive exonuclease activity at 37°C. 

6. The T4 polynucleotide kinase works well at room temperature. 

7. Although the PCR products purified directly by alcohol precipitation after end repairing 
are sufficient for routine cloning, passing the repaired PCR product mixtures through a 
gel filtration column prior to the alcohol precipitation can greatly enhance the cloning 
efficiency. 

8. In the ligation reaction, we routinely used 1:1 molar ratio between vector (dephosphory- 
lated) and insert. 



724 Wang 

References 

1. Clark, J. M. (1988) Novel non-templated nucleotide addition reactions catalyzed by pro- 
caryotic and eucaryotic DNA polymerases. Nucl. Acids Res. 16, 9677-9686. 

2. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, 
N. (1985) Primer-directed enzymatic amplification of DNA with a thermostable DNA 
polymerase. Science 230, 1350-1354. 

3. Scharf, S. (1990) PCR Protocols: A Guide to Methods and Applications. Academic, San 
Diego, CA. 

4. Jung, V., Pestka, S. B., and Pestka, S. (1990) Efficient cloning of PCR generated DNA 
containing terminal restriction endonuclease recognition sites. Nucl. Acids Res. 18, 6156. 

5. Mead, D. A., Pey, N. K., Herrnstadt, C, Marcil, R. A., and Smith, L. M. (1991) A univer- 
sal method for the direct cloning of PCR amplified nucleic acid. Biol technology 9, 657. 

6. Kovalic, D., Kwak, J., and Weisblum, B. (1991) General method for direct cloning of 
DNA fragments generated by the polymerase chain reaction. Nucl. Acids Res. 19, 4560. 

7. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. (1991) Construction of T-vectors, a 
rapid and general system for direct cloning of unmodified PCR products. Nucl. Acids Res. 
19, 1154. 

8. Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1989) Molecular Cloning: A Labora- 
tory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 



14 

Enzyme-Free Cloning of PCR Products 
and Fusion Protein Expression 

Brett A. Neilan and Daniel Tillett 
1. Introduction 

Numerous techniques have been developed for the cloning of polymerase chain reac- 
tion (PCR) products. These include the incorporation of restriction enzyme sites into the 
PCR primers (7), blunt-end cloning (2,3), TA cloning (4,5), ligation-independent clon- 
ing (LIC) (6-11), and in vivo cloning (12,13). Because these methods are effective, they 
all require either extensive enzymatic treatment of the PCR product or vector (1,4- 
11,14), the use of PCR primers containing nonstandard bases (9,10), or specialized vec- 
tors or bacterial strains (2,13,15). In addition, cloning into specialized expression or 
reporter vectors requires multiple subcloning steps. Under most circumstances, these 
limitations pose little difficulty, however, under certain conditions the current techniques 
can be severely limiting. For example, the direct cloning of a PCR product into a particu- 
lar site of an unusual expression vector is problematic. This can prove especially com- 
plex if the vector lacks suitable cloning sites, or a genetic basis for insert screening. 

Recently, a novel technique to clone PCR products, termed "heterostagger PCR clon- 
ing," was introduced (16). This technique involves the generation of two related PCR 
products by the design of two sets of primers. The two sets are identical except the 
second primer set contains a three-base guanosine five prime tail (e.g., first set 5'-TAT..., 
second set 5'-GGGTAT...). Two PCR reactions are performed using the first untailed- 
forward primer and the tailed-reverse primer, and then the tailed-forward primer with 
the untailed-reverse primer. This produces two PCR products that, when mixed, dena- 
tured, and allowed to reanneal, create fragments containing 3' CCC overhangs that are 
cloned by ligation to a vector containing 3' GGG overhangs. Although elegant, this tech- 
nique is limited by the need for extensive vector preparation, and the requirement for a 
unique restriction enzyme site at the desired cloning location within the chosen vector. 

We reasoned that if the required 3 bp tail was increased to 12-18 bp, then the resulting 
"heterostaggered" PCR products could be cloned using the LIC procedure (6). In addition, 
by PCR amplifying the vector using a compatible set of tailed and nontailed primers, the 
PCR products could be cloned without further enzymatic reaction (see Fig. 1). 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

125 



126 



Neilan and Tillett 




PCR 




insert 




Pool 




i 



Denature & re-anneal 



5 'overhangs 



3 'overhangs 




Nicked circular 
products 



5' 

mm* 



5' 



5' 



Linear products 

Fig. 1. The enzyme-free cloning strategy. Four PCR reactions are performed, two vector 
and two insert: the first vector PCR is performed with the 5' vector-short and the 3' vector-long 
(tailed) primers, whereas the second vector PCR uses the 5' vector-long and the 3' vector-short 
primers; the first insert PCR uses the 5' insert-short and 3' insert-long primers, whereas the 
second is performed using the 5' insert-long and the 3' insert-short primers. The long primers 



Enzyme-Free Cloning 127 

The enzyme-free approach has a number of advantages over other cloning meth- 
ods. First, no post-PCR enzyme reactions are required, such as restriction enzyme 
digestions, phosphatase treatments, partial DNase digestions, or ligations, and thus the 
potential for cloning failure is reduced. Second, the approach is simple and requires 
minimal sample handling. Third, the enzyme-free procedure enables the PCR insert to 
be cloned directly into a desired plasmid locus without the need for a suitable restric- 
tion enzyme site. This feature is particularly useful for ensuring a DNA fragment is 
directionally cloned into the correct reading frame for protein expression or for the 
creation of fusion proteins. In addition, the technique can be used to easily introduce a 
short amino acid tag sequence into either the amino or carboxy terminus of a protein. 

2. Materials 

2. 1. DNA Amplification Reagents 

It is advisable to store small aliquots of these solutions at 4°C for short periods (up 
to 3 wk). Stock solutions should be kept at -20 C C for longer periods of storage. Pref- 
erably, stock solutions are kept as small aliquots to reduce deterioration of quality 
resulting from repeated freeze-thaw cycles. All solutions are made using deionized 
and autoclaved water. The pH values are for solutions at 25°C. Many suppliers of 
thermostable polymerases also supply the other PCR reagents. 

1. 10X polymerase buffer: 200 mM Tris-HCl (pH 8.2), 100 mM KC1, 100 mtf (NH 4 ) 2 S0 4 , 
1.0% Triton X-100, and 1 mg/mL nuclease-free bovine serum albumin (BSA). 

2. 2 mM deoxynucleotide 5'-triphosphate (dNTP) mix: combine equal volumes of 8 mM 
deoxyadenosine 5 '-triphosphate (dATP), 8 mM deoxycytidine 5 '-triphosphate (dCTP), 
8 mM deoxyguanosine 5'-triphosphate (dGTP), and 8 mM deoxythymidine 5'-triphos- 
phate (dTTP) (Boehringer). 

3. 25 mM MgCl 2 : solution is filter (0.22 urn) sterilized. 

4. Taq/Pfu DNA polymerase mix: Two enzymes combined in 10:1 ratio. Taq supplied by 
Fischer Biotech (Perth, Australia) and Pfu from Promega (Madison, WI). 

5. Oligonucleotide primers: available commercially and working solutions adjusted to 
10 pmol/uL in ddH,0 (see Note 3). 

2.2 Amplicon Purification, Hybridization, 
and Transformation Reagents 

1. 3.0 M sodium acetate: solution is filter (0.22 urn) sterilized and the pH is not adjusted. 

2. 80% ethanol: 80% (v/v) analytical-grade ethanol in ddH 2 0. 

3. Hybridization buffer: 100 mM NaCl, 10 mM Tris-HCl (pH7.4), 1 mM ethylenediamine 
tetraacetic acid (EDTA) (pH 8.0). 

4. Luria broth: 10 g Tryptone, 10 g NaCl, and 5 g Yeast Extract per liter of ddH 2 0. 



Fig. 1. (continued) differ from the short primers by possessing a 12 to 15 bp 5' tail, with each of 
the two nonhomologous insert primer tails complementary to only one of the two vector primer 
tails, thus ensuring the directional cloning of the desired fragment. Equimolar volumes of the 
four PCR products are mixed, heat-denatured, and allowed to reanneal. Eight annealing prod- 
ucts of equal probability are created, of which four contain either 5' or 3' 12-18 bp overhangs. 
Low-temperature annealing (25°C) allows these four products to form two nicked circular prod- 
ucts which can be transformed without ligation (see Note 8). Figure reproduced and modified 
from (19). 



128 Neilan and Til left 

Table 1 

Oligonucleotide Primers Described in this Chapter 

Primer Sequence (5' to 3') 

NMLF CATCACCATCACCATCAC-CAAGAAGGGGAACTCTTG 

NMSF CAAGAAGGGGAACTCTTG 

NMLR TGTAAAACGACGGCCAGT -TTATTGGGGAATCAGTTTAC 

NMSR TTATTGGGGAATCAGTTTAC 

PHL GrGArGGTGArGGrGArG-CATAGCTGTTTCCTGTGTG 

PHS CATAGCTGTTTCCTGTGTG 

PEL ACTGGCCGTCGTTTTACA -ACGTCGTGACTGGGAAAAC 

PES ACGTCGTGACTGGGAAAAC 

Dashes indicate the point of extension between short and long. Underlined and itali- 
cized sequences are the reverse complements for amplicon hybridization at the 3' and 5' 
ends, respectively, for the N-methyltransferase insert to be cloned (see Note 6). This table 
describes vector primers which encode a polyhistidine tag for fusion protein construction 
(PHL and NMSF). Alternatively, if such a tag is not required, an arbitrary sequence of 12- 
18 bp may be used to extend the short template specific primers sequences (as seen in the 
downstream primers, NMLR and PEL, used in this study). 



3. Methods 

3.1. Insert PC Rs 

Two insert reaction mixes are prepared and amplified. Each PCR mix contains a 
different insert short primer and insert long primer (see Fig. 1 and Table 1). 

1. Prepare genomic DNA as per standard protocols for the organism under investigation 
(see Note 1). 

2. In two thin-walled 200-[xL microfuge tubes combine: 2 (L of 10X polymerase buffer, 
2 \iL of 2 raM dNTP mix, 2 (xL of 25 mM, 1 iiL of genomic DNA (approx 10-100 ng), 
0.2 fxL of high fidelity Taq/Pfu polymerase mix (see Note 2), and 10.8 fxL of ddH 2 0. 
Keep these insert reaction mixes on ice. 

3. To one tube containing the insert reaction mix, add 1 uL of insert short-forward primer 
and 1 uL of insert long-reverse primer. To the other insert reaction mix, add 1 \ih of insert 
long-forward primer and 1 (xL of insert short-reverse primer. 

4. Cover each reaction with mineral oil unless a thermocycler with a heated lid is available. 
Subject each tube to an initial denaturation of 2 min at 94 C C in a thermal cycler. Incubate 
tubes with insert PCRs for 26 cycles of 94°C, 10 s; primer annealing temperature for 20 s; 
and 72°C for 90 s; followed by a final extension at 72°C for 5 min. PCR annealing tem- 
perature and extension times may vary depending on the primer design and the size and 
structure of the target gene (see Notes 2 and 4). 

5. Determine that a single PCR amplicon of expected size has been generated for each insert 
PCR by agarose/TAE gel electrophoresis (17). 

3.2. Vector PCRs 

Two vector PCR mixes are prepared and amplified. Each PCR mix contains a dif- 
ferent vector short primer and vector long primer (see Fig. 1 and Table 1). 



Enzyme-Free Cloning 129 

1. Prepare plasmid DNA as per standard protocols (17) (see Note 5). 

2. In two thin-walled 200-uT microfuge tubes combine: 2 u.L of 10X polymerase buffer, 
2 uL of 2 mM dNTP mix, 2 (xL of 25 mM, 1 uL of plasmid DNA (approx 1-10 pg), 0.2 uL 
of high-fidelity Taq/Pfu polymerase mix, and 10.8 |xL of ddH 2 0. Keep these vector reac- 
tion mixes on ice. 

3. To one tube containing the vector reaction mix, add 1 u,L of vector short-forward primer 
and 1 u,L of vector long-reverse primer. To the other vector reaction mix, add 1 [xL of 
vector long-forward primer and 1 u,L of vector short-reverse primer. For experiments 
requiring the heterologous expression of a fusion protein the vector primers are 
designed to incorporate the sequence encoding a 6 histidine residue tag (see Note 6 
and Table 1). 

4. Subject each tube to an initial denaturation of 2 min at 94°C in a thermal cyler. Incubate 
tubes with vector PCRs for 26 cycles of 94°C, 10 s; primer annealing temperature for 
20 s; and 72°C for 90 s; followed by a final extension at 72°C for 5 min. PCR annealing 
temperature and extension times may vary depending on the primer design and the size 
and structure of the cloning vector (see Notes 2 and 4). 

5. Determine that a single PCR amplicon of expected size has been generated for each vec- 
tor PCR by agarose/TAE gel electrophoresis (17). 

3.3. Purification of Insert and Vector Amplicons 

Although not essential, it is advisable for increased cloning efficiency to purify PCR 
products from other PCR components, including polymerases and oligonucleotides. 

1. In one 1.5-mL centrifuge tube combine the two insert PCR products. In another tube, 
combine both vector PCRs. To each tube, add one-tenth volume of 3 M sodium acetate 
and 2 volumes of 80% ethanol. Alternatively, commercially available kits for the purifi- 
cation of PCR products may be employed, following the manufacturers instructions. 

2. It is necessary at this stage to determine the concentration of each purified PCR product 
mix by either ultraviolet (UV) spectrometry (260 nm) or by ethidium bromide staining 
against known DNA standards. 

3.4. Hybridization of Insert and Vector Amplicons 

1. In a single 200-uL microfuge tube combine purified insert and vector amplicons at an 
equimolar ratio. Approximately 50 ng of a 3 kb vector amplicon is required and the 
amount of insert added accordingly (e.g., 5 ng of a 300 bp insert PCR fragment). 

2. This volume is lyophilized and resuspended in 50 |xL of hybridization buffer prior to 
denaturation and hybridization. A cloning control reaction, containing only 50 ng of vec- 
tor PCRs in hybrdization buffer should also be prepared in a microfuge tube. 

3. Both test (containing insert and vector fragments) and control (vector only) hybridization 
reactions are then heated to 95°C for 3 min and thermal cycled at 65°C for 2 min and 
25°C for 15 min for a total of 4 cycles. This cycling process is essential to the success of 
the cloning procedure. 

3.5. Transformation of Hybridized PCR Amplicons 

1. Approx 2 u.L of the hybridization reactions are added to 150 u,L of chemically (CaCl 2 ) 
prepared competent Escherichia coli (E. coli) DH5ct cells (17). 

2. The PCR product/cell mix is allowed to transform on ice for 30 min followed by heat 
shock for 2 min at 42°C. 



130 Neilan and Til left 

3. Cells are allowed to recover by incubation in Luria broth for 1 h, shaking at 37°C. An 
appropriate plasmid-borne antibiotic resistance marker is then challenged for transformant 
selection. Followed by screening for positive clones by colony PCR using both of the 
short-insert primers in an amplification reaction as detailed above (see Subheading 3.1. 
and Notes 5 and 7). 

4. Notes 

1 . One benefit of this method is that the quality of genomic and plasmid DNA need not be as 
high as that required for successive restriction endonuclease and ligation reactions used 
in non-PCR cloning procedures. 

2. The PCR annealing temperature is calculated for only the region that is target specific and 
would hybridize to template sequence in the first amplification cycle. 

3. This approach requires that a number of primers (eight) are synthesized. However, the 
advantages offered by this technique outweigh the added cost, especially given the low 
cost of commercial oligonucleotides. It was found for many applications that a common 
set of vector primers can be used and thus only the two tailed insert primers are required 
over other regular PCR cloning procedures. 

4. This approach requires that both the vector and insert are able to be amplified by PCR. In 
most circumstance this is not an important limitation. Zhang et al. (15) have recently intro- 
duced a PCR-based cloning procedure based on homologous recombination (termed ET 
cloning) able to overcome this limitation. However, this procedure requires two rounds of 
transformation and selection and either the use of specialized E. coli strains (sbcA), or the 
use of a second modifying plasmid (pBAD-ETX). While the ET cloning procedure is supe- 
rior for some applications (e.g., the engineering of very large BAC clones), the simplicity 
of the described enzyme-free procedure suggests it will prove generally advantageous. 

5. As an alternative to purifying plasmid DNA a single bacterial colony containing the 
required cloning vector may be used in a colony PCR. If this is the case, the level of 
ddH 2 in the PCR should be adjusted to 11.8 uL. 

6. The NMT primers, NMLF, NMSF, NMLR and NMSR (see Table 1), were designed to 
amplify the N-methyltransferase (NMT) region of the microcystin synthetase gene 
mcyA. The forward long primer, NMLF, was designed to incorporate an amino terminal 
6 histidine (6X His) purification tag into both expressed proteins. The pUC19 primers 
PHL, PHS, PEL, and PES (see Table 1), were designed to allow the regulated expres- 
sion of the 6X His tagged NMT gene from the pUC19 lac promoter. Four parallel PCR 
reactions were performed with the corresponding long and short primer pairs to amplify 
the pUC19 vector, together with the NMT gene region from the cyanobacterium 
Microcystis aeruginosa PCC7806. The two NMT amplifications were performed using 
the two primer pairs NMLF/NMRS and NMSF/NMLR. The denaturation and hybrid- 
ization reaction was performed with 40 ng of the pUC 19 pooled PCR products and 15 ng 
of the NMT (1.2 kb) pooled PCR products in 50 uL of hybridization buffer as described 
previously. Two microliters of the reaction was used to transform CaCl 2 competent 
cells of E. coli DH5a. Transformation of E. coli DH5a cells with the NMT PCR prod- 
ucts resulted in 44 colonies. Eight colonies were selected from the NMT transformation 
and checked by PCR for the appropriate cloned insert DNA using the NMT short prim- 
ers NMSF/NMSR. Five of the eight plasmids examined were found to contain the cor- 
rect NMT fragment. A number of individual clones were sequenced to ensure mutations 



Enzyme-Free Cloning 131 



(Didiuns) 



36 4&— 

26625— 
20 040— 



N 
O 



Fig. 2. Sodium dodecylsulfate-polyacrylamide gel electrophoreses (SDS-PAGE) analysis 
of purified recombinant N-methyltransferase (see Note 6) and O-methyltransferase protein frag- 
ments from M. aeuginosa PCC7806 overexpressed in E. coli BL21 using the described enzyme- 
free cloning method. The 8% polyacrylamide gel was stained with Coomassie blue. The masses 
of the markers are indicated at the left of the gel. Lane 1 : molecular size marker (New England 
BioLabs), Lane 2: affinity column purified fraction of N-methyltransferase, Lane 3: affinity 
column purified O-methyltransferase from M. aeruginosa PCC7806. 



had not been introduced into cloned sequences. Mutation-free clones of the NMT 
(pNMG) were selected and transformed into the E. coli expression strain BL21 (18). 
These clones were successfully used to over-express and purify these two proteins in 
E. coliBL2l (see Fig. 2). 

7. The enzyme-free procedure provided a highly efficient means for cloning PCR products 
independent of vector restriction enzyme sites. Theoretically, 50% of the PCR products 
should be clonable, that is, all molecules with either 5' or 3' overhangs. In practice, trans- 
formation efficiencies of up to 8X 10 4 /}j.g of insert DNA were obtained using standard 
CaCl 2 competent cells (10 6 /ng of pUC19 plasmid). The use of a high-fidelity PCR mix 
minimizes the introduction of PCR-derived mutations, both within the insert and vector. 
In addition, no colonies were obtained from the transformation of the control PCR ampli- 
fied linear vector hybridization reaction. 

8. PCR yield and efficiency is typically greater when applied to noncircularized templates. 
We have seen greater cloning efficiencies when the plasmid vector is linearized prior to 
vector amplifications. This step is not usually necessary given that only one correct clone 
is required in many experiments. 



132 Neilan and Til left 

References 

1. Scharf, S. J., Horn, G. T., and Erlich, H. A. (1986) Direct cloning and sequencing analysis 
of enzymatically amplified genomic sequences. Science 233, 1076-1078. 

2. Costa, G. L., Grafsky, A., and Weiner, M. P. (1994) Cloning and analysis of PCR-gener- 
ated DNA fragments. PCR Meth. Appl. 3, 338-345. 

3. Scharf, S. J. (1990) Cloning with PCR, in PCR Protocols, Innis, M. A., Gelfand, D. H., 
Sninsky, J. J., and White, T. J., eds. Academic, New York, pp. 84-91. 

4. Holton, T. A. and Graham, M. W. (1991) A simple and efficient method for direct cloning 
of PCR products using ddT-tailed vectors. Nucl. Acids Res. 19, 1 156. 

5. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Construction of T-vec- 
tors, a rapid and general system for direct cloning of unmodified PCR products. Nucl. 
Acids Res. 19, 1154. 

6. Aslanidis, C. and de Jong, P. J. (1990) Ligation-independent cloning of PCR products 
(LIC-PCR). Nucl. Acids Res. 20, 6069-6074. 

7. Aslanidis, C, de Jong, P. J., and Schmitz, G. (1994) Minimal length requirement of the 
single-stranded tails for ligation-independent cloning (LIC) of PCR products. PCR Meth. 
Appl. 4, 172-177. 

8. Hsiao, K.-C. (1993) Exonuclease III induced ligase-free directional subcloning of PCR 
products. Nucl. Acids Res. 21, 5528-5529. 

9. Kaluz, S. and Flint, A. P. F. (1994) Ligation-independent cloning of PCR products with 
primers containing non-base residues. Nucl. Acids Res. 22, 4845. 

10. Nisson, P., Rashtchian, A., and Watkins, P. (1991) Rapid and efficient cloning of Alu- 
PCR products using uracil DNA glycosylase. PCR Meth. Appl. 1, 120-123. 

11. Yang, Y.-S., Watson, W. J., Tucker, P. W., and Capra, J. D. (1993) Construction of recom- 
binant DNA by exonuclease recession. Nucl. Acids Res. 21, 1889-1993. 

12. Bubeck, P., Winkler, M., and Bautsch, W. (1993) Rapid cloning by homologous recombi- 
nation in vivo. Nucl. Acids Res. 21, 3601-3602. 

13. Oliner, J. D., Kinzer, K. W., and Vogelstein, B. (1993) In vivo cloning of PCR products in 
E.coli. Nucl. Acids Res. 21, 5192-5197. 

14. Shuldiner, A. R., Scott, L. A., and Roth, J. (1990) PCR-induced (ligase-free) subcloning: 
a rapid reliable method to subclone polymerase chain reaction (PCR) products. Nucl. Acids 
Res. 18, 1920. 

15. Zhang, Y., Buchholz, F., Muyrers, J. P. P., and Stewart, A. F. (1998) A new logic for DNA 
engineering using recombination in Escherichia coll. Nature Gen. 20, 123-128. 

16. Liu, Z. (1996) Hetero-stagger cloning: efficient and rapid cloning of PCR products. Nucl. 
Acids Res. 24, 2458-2459. 

17. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, a Laboratory 
Manual, 2nd ed. Cold Spring HarborLaboratory Press, NY. 

18. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Use of T7 
RNA polymerase to direct expression of cloned genes. Meth. Enzymol. 185, 60-89. 

19. Tillett, D. and Neilan, B. A. (1999) Enzyme free cloning: a rapid method to clone PCR 
products independent of vector restriction enzyme sites. Nucl. Acids Res. 27, e26. 



15 



Directional Restriction Site-Free Insertion 
of PCR Products into Vectors 

Guo Jun Chen 
1. Introduction 

The polymerase chain reaction (PCR) technique has proved to be a powerful tool 
for rapid amplification of DNA fragments of interest during cloning. Insertion of PCR 
products into suitable vectors in order to construct plasmids for protein expression, or 
to create chimeric genes or to study interactions between proteins and DNA requires a 
method that allows the precise insertion of a DNA fragment at a defined position in the 
vector without altering the surrounding DNA sequence. The commonly used cloning 
methods, such as blunt-end insertion (1,2), introduction of restriction sites on both 
ends of the PCR products (3,4), T vectors (5) and ligation-independent cloning (6,7) 
do not usually meet the needs because of the limited cloning sites available on the 
vector and/or the necessity of the nucleotide change. Hence, the cloning process may 
become very complicated to insert a PCR product into a defined location on the vector 
without the change of the nucleotides. 

A high-efficiency restriction site-free cloning method (8) has been developed (see 
Fig. 1). It can precisely insert a DNA fragment of interest directionally into a vector at 
desired location without altering any nucleotide on either the DNA fragment or the 
vector. This cloning method uses a pair of primers to link two DNA molecules (insert 
and vector) at a precise junction to form a new double-stranded plasmid. The primer 
can functionally be divided into two parts: 3' portion and 5' portion. The 3' portion 
is used to amplify the DNA fragment of interest by PCR. The DNA sequence of this 
portion is thus homologous to the DNA insert at the junction. The 5' portion is 
used to generate a PCR product in which the DNA fragment is flanked on both sides 
by a piece of DNA that is homologous to the vector at the junction. In the thermal-cycling 
elongation (TCE) step, both the 3' end and the 5' end of each single-stranded DNA of 
the PCR product will anneal to their complementary strand of the vector. The 3' end 
will be extended by DNA polymerase using the vector as a template. This results in 
a fusion between the DNA fragment and the vector. At the end of the TCE reaction, 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

133 



134 



1. Design of primer. 



5' -3' 3- E 



2. Amplification of insert 



I 



I 



3. Fusion of insert and vet 



Chen 




i 



4. Selection of plasmid. 



Fig. 1. Scheme of restriction site-free cloning method. 



Restriction Site-Free Cloning 135 

the newly synthesized single-stranded DNAs will anneal to their complementary 
strands to form stable nicked double-stranded plasmids. Dpnl endonuclease, which 
specifically cuts double-stranded methylated and hemimethylated 5' -Gm 6 ATC- 3' 
DNA sequences (9), is used to selectively digest the methylated parental DNA tem- 
plate. The enriched preparation of the plasmid is then used for transformation. The 
following is an application of this method to construct an IPTG inducible expression 
plasmid that produces wild type Echerichia coli (E. coli) peptidyl-tRNA hydrolase. 

2. Materials 

2.1. Enzymes and Reagents 

1. Expand High Fidelity PCR System (Roche Molecular Biochemicals). Store at -20 °C. 

2. P/uTurbo™ DNA polymerase (Stratagene). Store at -20°C. 

3. Deoxynucleotide triphosphates (dNTPs) (Roche Molecular Biochemicals). Store at -20°C. 

4. E. coli BL 21 chromosomal DNA [according to supplied protocol in QIAamp DNA Mini 
Kit (Qiagen)]. Store at -80°C. 

5. 10X DNA loading buffer: 0.4% bromophenol blue, 0.4% xylene cyanol, 50% glycerol. 
Store at 4°C. 

6. TAE agarose gels. Store at 4°C. 

7. IX TAE running buffer: 50X TAE: 242 g Tris base, 57.1 mL glacial acetic acid, 100 mL 
0.5 M ethylenediamine tetraacetic acid (EDTA), H 2 to 1000 mL. Store at room temperature. 

8. Ethidium bromide solution: dissolve 50 mg of ethidium bromide in 100 mL of H 2 0. Use 
diluted 1:1000. Store at 4°C and protect from light. 

9. QIAquick Gel Extraction Kit (Qiagen). Store at room temperature. 

10. Vector pDS56/RBSII. (Caution: Vectors should be isolated from dam + E. coli stains. Plas- 
mid from dam' strains is not suitable for this method.). Store at -80°C. 

1 1. Dpnl restriction enzyme (Roche Molecular Biochemicals). Store at -20°C. 

12. Electroporation competent cells. (Wash cells in log phase (OD 600 = 0.6-0.8) with 100X 
cell pellet volume ice cold sterile distilled water or 5-10% glycerol twice by centrifuga- 
tion at approx 5000 g. Resuspended the cell pellet in 5X volume 10% glycerol solution.) 
Store at -80°C. 

13. SOC medium: 2% tryptone, 0.5% yeast extract, 0.05% NaCl and 20 mM glucose. 
Store at 4°C. 

14. Luria Bertani (LB) ampicillin agar plates. Store at 4°C. 

15. LB medium: 1% tryptone, 0.5% yeast extract, 0.5% NaCl. Store at 4°C. 

2.2. Equipment 

1. Centrifuge (up to 14,000g). 

2. 1 .5-mL Eppendorf tubes. 

3. Thermal-cycler and 0.5-mL Eppendorf tubes. 

4. DNA gel box and power supply. 

5. Electroporation apparatus and 0.2-cm electroporation cuvets. 

6. 37°C Incubator. 

7. Water bath (37°C and 65°C). 

8. Ultraviolet (UV) light box. 



Table 1 

Sample Set for Cloning (see Subheading 3.1.) 



Primer 5' Portion sequence 3' Portion sequence 

forward TCACACAGAATTCATTAAAGAGGAGAAATTAACT ATGACGATTAAATTGATTGTCGGCC 
reverse CCCCAGGCGTTTAAGGGCACCAATAACTGCC TTATTGCGCTTTAAAGGCGTGCAAT 



3. Methods 

3. 1. Primer Design 

1. Draw a resulting plasmid map by inserting the DNA fragment into the desired position in 
the vector. At the junction of the two DNA molecules, select a sequence (at least 30 bases 
long) from the vector for the 5' portion sequence of the primer and a sequence (at least 20 
bases long) from the DNA fragment for the 3' portion. The selected sequences for the 
5' portion and the 3' portion should give a predicted melting temperature (T m ) around 
60-70°C and 50-60 c C, respectively. (The commonly used primer Tm estimation formula 
is T m (°C) = 2 x [total number of A and T] + 4 x [total number of G and CJ.) The 5' portions 
or the 3' portions of forward primer and reverse primer should have a similar T m . Ensure 
that both forward primers and reverse primers have the right orientation. They should 
always have the DNA sequence in the 5' to 3' direction from the vector to the DNA frag- 
ment (see Note 2). 

Table 1 shows a sample set of primers being successfully used for cloning wild-type 
E. coli peptidyl-tRNA hydrolase to pDS56/RBSII (8): 

3.2. Amplification of the Peptidyl-tRNA Hydrolase Encoding Region 

1. Prepare a 100-uE PCR mixture in a 0.5-mL Eppendorf tube (on ice) as indicated as 
follows. 

a. IX PCR reaction buffer with Mg 2+ (supplied with Expand High Fidelity PCR System). 

b. 0.5 (xg E. coli BL21 strain chromosomal DNA. 

c. 0.5 \iM forward primer. 

d. 0.5 u.M reverse primer. 

e. 200 uM dNTPs each. 

f. 2.5 U Expand High Fidelity DNA polymerase (add last, see Note 3). 

g. Amount of fresh redistilled H 2 to bring the final volume up to 100 (xL (add first). 

2. Thoroughly mix and then centrifuge briefly to bring the sample to the bottom of the tube. 

3. Set a 25-cycle two-step PCR program on a thermal-cycler. The denaturation is at 95°C for 
30 s and the annealing/elongation is at 68 C C for 1 min with an extra 10 min at 72°C at the 
end of the reaction. Then hold the reaction at 4°C (see Note 4). 

4. Place the Eppendorf tube in the thermal-cycler and turn on the top heating cover. If the 
thermal-cycler does not have one, carefully add 50 \iL mineral oil to overlay the reaction 
mixture to prevent evaporation. Then start the PCR program. 

5. Mix the PCR product with 10 jiL 10X DNA loading buffer. Load the sample on a 1% 
agarose TAE gel. Beware to connect the electrodes of the gel box in correctly to the 
power supply. Electrophoresis at 5 V/cm in IX TAE buffer containing 0.5 (ig/mL 
ethidium bromide until the dye markers have migrated an appropriate distance. (Caution: 
Ethidium bromide is potent carcinogen. Wear glove to handle it.) (See Note 5). 

6. Excise the expected band from the TAE gel on UV light box as quickly as possible. UV 
light will damage the DNA. (Caution: UV light induces DNA mutagenesis and is harmful 
to naked eyes. Wear protection glasses and/or a shield.) (See Note 6). 



Restriction Site-Free Cloning 137 

7. Extract the DNA fragment from the gel slice according to the protocol supplied in the 
QIAquick Gel Extraction Kit (Qiagen). 

3.3. Insertion of PCR Product Into Vector 

1. Quantify both the vector and the purified PCR product on an agarose gel. The amount of 
the DNA is estimated according to the known quantity of the marker. The vector should 
first be linearized by restriction enzyme digestion before applied to the agarose gel for 
electrophoresis. The running conditions are the same as that in Subheading 3.2., step 5 
(see Note 7). 

2. Set up a 25-u.L TCE reaction mixture as following: 

a. IX PCR reaction buffer (supplied with PfuTurbo™ DNA polymerase). 

b. 5-10 ng pDS56/RBSII. 

c. 50-100 ng purified PCR products described above (see Note 8). 

d. 200 \iM dNTPs each. 

e. 1 U PfuTurbo™ DNA polymerase (add last, see Note 9). 

f. Amount of fresh redistilled H-,0 to the final volume of 25 fxL (add first). 

3. The thermal-cycle program is 95 C C 2 min for denaturation and 68°C 10 min for anneal- 
ing/elongation with 20 cycles. Hold the reaction at 4°C (see Note 4). 

4. Place sample in a thermal-cycler and start the TCE program. 

3.4. Dpn/ Digestion and Transformation 

1. After TCE reaction, add 5 U of Dpnl restriction enzyme directly to the TCE reaction 
mixture. Mix thoroughly and digest for 1-2 h at 37°C (see Note 10). 

2. Add 1-2 \iL Dpnl treated TCE reaction mixture to ice-cold 50 u,L E. coli Sure 2 
electroporation competent cells in a 1.5-mL Eppendorf tube (see Note 11). 

3. Gently and thoroughly mix and transfer to the bottom of ice-cold Bio-Rad 0.2-cm 
electroporation cuvet. Be sure the cell droplet contacts both electrodes and is free from air 
bubbles. 

4. Put the cuvet in a BioRad Gene Pulser™ chamber and perform electroporation with a 
setting of voltage at 2500 V, resistance at 200 Q, and capacitance at 25 \iF. (Time con- 
stants of approx 5 ms are usually obtained.) 

5. After electroporation, immediately add 450 \\L SOC medium (prewarmed to room tem- 
perature) to the electroporation cuvet and gently mix (see Note 12). 

6. Transfer the above mixture to a 1 .5-mL Eppendorf tube and put in a 37°C water bath for 
30 min. 

7. Plate 100 u,L of the transformation culture onto an LB agar plate containing 100 jig/mL 
ampicillin (see Note 13). 

8. Incubate the transformation plate upside down at 37°C overnight (12-16 h). 

9. Check the plate and pick up single colonies for further analysis. 

4. Notes 

1. The method described above has also successfully been applied to mutagenesis (8). In 
such a case, the primer is divided into three functional parts: a 5' portion, a mutation- 
generating portion and a 3' portion. The mutation-generating portion is flanked by the 5' 
portion and the 3' portion. Changing the nucleotide(s) in this portion can introduce muta- 
tions such as substitution, insertion, or deletion in the plasmid. If both forward primer and 
reverse primer are used to introduce mutations, it can simultaneously mutate two posi- 
tions that are far away from each other. 



138 Chen 

2. The length of the primer should be determined by the melting temperature (T m ) of the 5' 
portion and the 3' portion. The synthesis of long primers is prone to errors, therefore 
choose a high-quality primer supplier. Another solution is to design the primer as short as 
possible. In some cases, part of the DNA sequence will overlap between the DNA frag- 
ment and the vector (e.g., ATG codon exists in both DNA fragment and vector). This part 
can be used for both the 5' portion and the 3' portion of the primer. For substitution mu- 
tagenesis, the primer can even be reduced to the length of the 3' portion. 

3. Expanding high-fidelity DNA polymerase gives high yield compared with Pfu DNA poly- 
merase and low error compared with Taq DNA polymerase. Errors generated during early 
PCR steps can be amplified to a dramatic extent. It is suggested to use more templates, 
less PCR cycles, and high-yield and high-fidelity DNA polymerases. 

4. A three-step program with a lower annealing temperature is also suitable for this task. 
High-annealing temperature usually yields a high-specificity product. Use the DNA exten- 
sion rate recommended by DNA polymerase producer to calculate the elongation time. 
The elongation time should be long enough to obtain a full length PCR product or TCE 
product. It is especially important in the TCE step to ensure the synthesis of a full-length 
single-stranded plasmid. 

5. Nonspecific PCR products will interfere with the TCE reaction and may generate 
unwanted plasmids because their ends have DNA sequences homologous to the insertion 
region of the vector. Removing these by-products through electrophoresis will increase 
the positive percentage. 

6. To reduce the DNA damage, use long wavelength UV light or put a thin glass plate 
between the UV light box and the gel to block some UV light. 

7. For more accurate estimation, apply two to three different amount of each sample and 
marker to the gel. Do not overexpose the film. 

8. The ratio of the molarity of the plasmid to the insert is quite important to get good results. 
The optimum molar ratio is influenced by the number of TCE cycles. With the TCE 
program described in this protocol, the molar ratio (the plasmid to the insert) of 1 to 50-100 
gives the best results for cloning of peptidyl-tRNA hydrolase. It is always a good start to 
fix the amount of the vector and vary the amount of the PCR product to find out the 
optimum condition. 

9. Theoretically, the thermally stable DNA polymerase used for this task should have 3'-5' 
exonuclease activity but should not have strand displacement replication function and 
5'-3' exonuclease activity. These will ensure that the DNA polymerase stops DNA syn- 
thesis right at the 5' end and no extra nucleotides are added to the 3' end of the PCR 
product. 

10. The purpose of Dpnl treatment is to reduce the background. As Dpnl cuts fully methy- 
lated dsDNA and hemimethylated dsDNA, the plasmid used in this method should be 
isolated from dam* E. coli strains. It may use other restriction systems such as the Kunkel 
method (10) for this task, in which the E. coli dut + ung + strain will in vivo eliminate the 
parental plasmid containing deoxyuracil after transformation. 

11. At this stage, the Dpnl treated DNA can also be transformed to the chemically treated 
competent cell following the protocol provided by the supplier of competent cells. 
DNA for electroporation must have a very low salt concentration. High ionic strength 
will cause arcing. Do not use more than 2 [xL Dpnl treated TCE reaction mixture for a 
single electroporation. In case more DNA sample for transformation is needed, salt can 
be removed by DNA precipitation being described as follows. 



Restriction Site-Free Cloning 139 

a. Add 2 \iL Pellet Paint (Novagen) to the sample followed by 2.5 uL 3 M Na-Acetate 
pH 5.2. Mix the sample briefly. Use of Pellet Paint Co-Precipitant instead of tRNA or 
collegen as a DNA carrier in DNA precipitation has two advantages: one is that it is 
easy to visualize the DNA pellet; and two is that all the steps can be carried out at 
room temperature. 

b. Add 50 [xL ethanol and mix thoroughly. 

c. Spin the sample in a microcentrifuge at 14,000-1 6,000g for 5 min. 

d. Remove the supernatant with a pipet. 

e. Wash the pink pellet by adding 500 uL of 70% ethanol. Centrifuge at 14,000-16,000g 
for 2 min. Remove the supernatant and air-dry the pellet. 

f. Resuspend the pink pellet in deionized water. 

12. Cells will die very quickly after electroporation if SOC medium is not added. Therefore, 
add SOC medium to the cuvet as soon as possible. 

13. Dry the agar plates (exposed upside down) at 37°C for 2-4 h just before use. The plate 
should be able to soak up to 0.5 mL of media when plating. Do not plate too many cells on 
a single plate. Sometimes it will mask small colonies on the plate. Try to use more plates 
if all the transformation samples need to be plated. 

References 

1. Bhat, G. J., Lodes, M. J., Myler, P. J., and Stuart, K. D. (1991) A simple method for 
cloning blunt ended DNA fragments. Nucl. Acids Res. 19, 398. 

2. Liu, Z. G. and Schwartz, L. M. (1992) An efficient method for blunt-end ligation of PCR 
products. Biotechniqu.es 12, 28. 

3. Karn, J., Brenner, S., Barnett, L., and Cesareni, G. (1980) Novel bacteriophage lambda 
cloning vector. Proc. Natl. Acad. Sci. USA 77, 5172-5176. 

4. Scharf, S. J., Horn, G. T., and Erlich, H. A. (1986) Direct cloning and sequence analysis of 
enzymatically amplified genomic sequences. Science 233, 1076-1078. 

5. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Construction of T-vec- 
tors, a rapid and general system for direct cloning of unmodified PCR products. Nucl. 
Acids Res. 19, 1154. 

6. Aslanidis, C. and de Jong, P. J. (1990) Ligation-independent cloning of PCR products 
(LIC-PCR). Nucl. Acids Res. 18, 6069-6074. 

7. Haun, R. S., Serventi, I. M., and Moss, J. (1992) Rapid, reliable ligation-independent clon- 
ing of PCR products using modified plasmid vectors. Biotechniques 13, 515-518. 

8. Chen, G. J., Qiu, N., Karrer, C, Caspers, P., and Page, M. G. P. (2000) Restriction site- 
free insertion of PCR products directionally into vectors. Biotechniques 28, 498-500, 
504-505. 

9. The NEB Transcript 1998, vol. 9 no. 1, page 7, GATC: Dpnl, DpnII, Mbol or Sau3A I. 
10. Ling, M. M. and Robinson, B. H. (1997) Approaches to DNA mutagenesis: an overview. 

Analyt. Biochem. 254, 157-178. 



16 

Autosticky PCR 

Directional Cloning of PCR Products with Preformed 5' Overhangs 

Jozsef Gal and Miklos Kalman 
1. Introduction 

The polymerase chain reaction (PCR) is a method of central importance in molecu- 
lar biology (1,2). The DNA fragment of interest is often amplified for cloning pur- 
poses. A frequently used experimental approach is to include extra restriction 
endonuclease cleavage sites in the amplification primers, digestion of the PCR prod- 
uct with the corresponding enzymes, and ligation of the product to a linearized cloning 
vector (3). However, the efficiency of cleavage by certain restriction endonucleases is 
rather low because of the cleavage site(s) being too close to the termini of a DNA 
fragment (4,5), and internal restriction sites of the fragment might also complicate the 
cloning. 

During PCR, the DNA fragment of interest is amplified with primer oligonucle- 
otides. Each strand of the amplified DNA fragment contains a built-in amplification 
primer in its 5' terminus. When a strand of the PCR product serves as a template for the 
amplifying DNA polymerase, the segment that is copied last is just the built-in primer. 

In nucleic acids, abasic sites are positions where the base is missing from the sugar- 
phosphate backbone. Abasic sites in a DNA template are noninstructional for a DNA 
polymerase, making it stall during synthesis of the complementary strand (6-11). 
Autosticky PCR (AS-PCR) products are amplified with primers containing abasic sites 
(10,11). First, the primer is incorporated into a PCR product strand. When this strand 
acts as template during further cycles, the amplifying polymerase is stalled at the 
primer-borne abasic position, resulting in the formation of single-stranded 5' overhangs 
on the termini of the AS-PCR product. The overhangs enable ligation of the AS-PCR 
product to a vector cut by the corresponding enzymes. 

A stable structural analog of 2'-deoxyribose, a tetrahydrofuran derivative, was cho- 
sen instead of naturally occurring abasic sites (7). It differs from the natural 2'-deox- 

*Patent pending No. P9801320 Hungarian Patent Office. 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

141 



142 Gal and Kalman 

yribose abasic site by having a hydrogen instead of a hydroxyl group on the 1' carbon 
of the deoxyribose ring. This difference makes tetrahydrofuran abasic sites highly 
resistant to the chemical conditions applied during conventional oligonucleotide syn- 
thesis based on phosphoramidite chemistry, enabling the incorporation of these sites 
into primers. The protected derivative of the tetrahydrofuran abasic site, ready for 
primer synthesis, is commercially available. 

The behavior of DNA polymerases at the terminus of a linear template (12,13) and 
at an abasic site proved to be similar. Pfu and Vent, which are proofreading poly- 
merases (14,15), are stalled before abasic sites without extra 3' nucleotide addition 
(9,11). Taq polymerase does not possess proofreading 3'-5' exonuclease activity 
(16,17), and adds an extra nucleotide, predominantly a dAMP residue, opposite an 
abasic site (8,10,11). Our results showed that in the ligation junction of the AS-PCR 
product and the vector, the abasic site should be opposed either by a polymerase- 
added extra nucleotide, or the 5' terminal residue of the vector-borne overhang (11). 
According to the aforementioned, the nontemplated 3' extra nucleotide addition 
activity of the desired polymerase should be taken into consideration when designing 
AS-PCR primers. 

In the resulting clones, the abasic position is either substituted by a nucleotide, 
frequently a dTMP residue, or is deleted. Rarely, the deletion of the abasic position is 
accompanied by a minor deletion in the vicinity of the position (10,11). 

The AS-PCR method has several advantages. It allows directional cloning of PCR 
products. Modification of the amplification product is unnecessary before ligation, so 
the end sensitivity of restriction enzymes (4,5) does not hamper cloning. Theoreti- 
cally, any desired 5' overhang can be generated, including overhangs that correspond 
to restriction sites present within the amplified sequence. A drawback of the method is 
that it is rather difficult to predict the result of the repair of the abasic positions. 

2. Materials (see Note 1) 

1. dSpacer, a protected tetrahydrofuran derivative phosphoramidite (7) and the so-called 
Chemical Phosphorylation Reagent (18) are commercially available (Glen Research, Ster- 
ling, VA). 

Both chemicals should be dissolved in acetonitrile (chemical DNA synthesis grade) 
and kept moisture-free. After they are dissolved, they should be used within 2-3 d, 1 wk 
maximum. The reagents might be taken back off the machine. For best results, seal them 
under argon. They should then be stored at -80°C, where the acetonitrile is frozen. Be 
very careful to keep them protected from moisture when the solutions are warming up. 

2. Vector DNA (preferably at 0.2-1 u,g/uL concentration). 

3. Restriction endonucleases with the corresponding 10X buffers. 

4. Sterile, deionized water, free from DNase and DNA. 

5. 100% (or 96%) and 70% ethanol. Keep at -20°C. 

6. 3 M Sodium acetate solution, pH 5.2. 

7. Phenol:chloroform:isoamylalcohol 25:24:1 mixture (19). 

8. Electrophoresis-grade agarose. 

9. IX TAE: 40 mM Tris-acetate, 1 mM ethylenediaminetetraacetic acid (EDTA) (19). 

10. 10 mg/mL ethidium bromide solution. Ethidium bromide is a powerful mutagen, so it 
should be used with caution. 



Autosticky PCR 143 

11. 6X gel loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% sucrose. 
Keep at 4°C. 

12. The QIAexII Gel Extraction Kit (QIAGEN, Valencia, CA) is recommended for the recov- 
ery of DNA after preparative gel electrophoresis. 

13. Taq DNA polymerase can be obtained from several providers, e.g., Invitrogen, 
Carlsbad, CA. Pfu DNA polymerase is available from Stratagene, La Jolla, CA. Vent 
DNA polymerase can be purchased from New England Biolabs, Beverly, CA. The 
commercially available DNA polymerases are usually provided with a 10X reaction 
buffer. Usually a solution of a Mg 2+ salt is also supplied by the manufacturer, if it is not 
contained within the 10X reaction buffer. 

In the lack of a manufacturer-provided buffer, the composition of 10X buffers are: 

a. Taq: 200 mM Tris-HCl (pH 8.4 at 25°C), 500 mM KC1. 

b. Pfu: 200 mM Tris-HCl (pH 8.75 at 25°C), 100 mM KC1, 100 mM (NH 4 ) 2 S0 4 , 1% 
Triton X-100, 1 mg/mL bovine serum albumin (BSA). 

c. Vent: 200 mM Tris-HCl (pH 8.8 at 25°C), 100 mM KC1, 100 mM (NH 4 ) 2 S0 4 , 1% 
Triton X-100. 

14. dNTP solutions are available from several commercial providers, e.g., Invitrogen. It is con- 
venient for further work to prepare a stock solution containing 5 mM of each dNTP. 

15. 10 \iM (10 pmol/uL) solution of the AS-PCR primers. 

16. Light mineral oil, if the PCR cycler operates without a heated lid. 

17. The QIAquick PCR Purification Kit (QIAGEN), or a similar kit is recommended for the 
purification of the AS-PCR product. 

18. T4 DNA ligase is available from several commercial providers, e.g., New England 
Biolabs. The enzyme is usually provided with a 10X concentrated buffer. If there is 
no manufacturer-provided 10X buffer, its recommended composition is 500 mM 
Tris-HCl (pH 7.5 at 25°C), 100 mM MgCL, 100 mM dithiothreitol (DTT), 10 mM 
rATP, 250 [ig/mL BSA. 

19. DH5ct Escherichia coli (E. coli) competent cells, either commercially available (e.g., 
from Invitrogen), or homemade (20). 

20. LB broth and LB/1.5% agar plates containing the corresponding antibiotic (19). 

21. Sol-1: 50 mM Tris-HCl, 10 mM EDTA, pH 8.0 at 25°C. 

22. Sol-2: 200 mM NaOH, 1% sodiumdodecyl sulfate (SDS). Best if freshly prepared 
before use. 

23. Sol-3: 3 M potassium acetate, pH 5.5. Chill on ice before use. 

24. Isopropanol. Keep at room temperature. 

25. TE solution: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 at 25°C. 

26. RNaseA (19), 100 (ig/mL solution (free from DNase). 

3. Methods 

3.1. Primer Design 

The design of the primers is a very important step of AS-PCR. It was found that 
there should be a nucleotide opposite the abasic site in the AS-PCR product-vector 
ligation junction (11). Without this nucleotide, AS-PCR products were difficult or 
impossible to clone. The opposing nucleotide can either be a polymerase-added extra 
nucleotide, or the 5' terminal nucleotide of the vector-borne overhang. According to 
the above, the primer design should consider the 3' extra nucleotide addition capacity 
of the used polymerase. 



144 Gal and Kalman 

Taq polymerase adds an extra nucleotide opposite abasic sites (8,10,11). It was also 
found that Taq polymerase can proceed a further nucleotide over the abasic position 
before it ceases synthesis (11), so it is advisable to contain a nucleotide between the 
restriction overhang and the abasic site, to provide "buffer" space for the read-through. 
This latter nucleotide can be exploited for the regeneration of the restriction site by 
means of the in vivo repair. Based on the above, AS-PCR primers designed for use 
with Taq polymerase should be arranged as: 5 '-restriction overhang-"buffer" nucle- 
otide-abasic position-template hybridizing segment-3'. For example, if the primer is 
designed for a BamHl site, 5'-P-GATC-C-S-template hybridizing segment-3', where 
"P" stands for a phosphate group, "S" for a tetrahydrofuran abasic site (see Fig. 1). 

Pfu and Vent polymerases do not add a nucleotide opposite the abasic postion when 
stalled (9,11). In this case, AS-PCR primers should be designed so as in the ligation 
junction between the AS-PCR product and the linearized vector, the 5' terminal nucle- 
otide of the vector-borne overhang is positioned opposite the abasic site. For example, 
if the primer is designed for aBamHI site, 5'-P-GAT-S-template hybridizing segment- 
s' (see Fig. 1). 

Double noninstructional sites in the template block DNA polymerases more effi- 
ciently than a single site (21). However, it is not recommended to use tandem abasic 
sites in AS-PCR primers, because it dramatically lowers the efficiency of cloning, 
probably because of the difficult repair situation (//). 

It is also very important to phosphorylate AS-PCR primers on their 5' termini. 
Dephosphorylation of AS-PCR products significantly decreased the efficiency of 
cloning (//). It is very important that the 5' overhangs of the AS-PCR product could 
be ligated. 

Only the template-hybridizing portion of AS-PCR primers should be taken into 
consideration when the annealing temperature is determined. For hybridizing segments 
around 20-25 nucleotides long, a well-working empirical formula is: 4X (number of 
G or C residues) + 2X (number of A or T residues) - 5 = recommended annealing 
temperature in °C. We suggest to design the hybridizing segment for about 55°C 
annealing temperature, based on the aforementioned formula. The general consider- 
ations of primer design (minimal secondary structure, minimal hybridization of the 
two primers with each other, no long homopolymeric segments, and so on) also apply 
to AS-PCR primer design. 

3.2. Primer Synthesis (see Note 2) 

Several companies that provide custom oligonucleotide synthesis (e.g., Sigma- 
Genosys, The Woodlands, TX, or New England Biolabs) offer the synthesis of tet- 
rahydrofuran abasic site-containing oligonucleotides. The chemical 5' phosphorylation 
of AS-PCR primers is strongly recommended, because enzymatic phosphorylation is 
generally less efficient (18). 

In case of having access to an oligonucleotide synthesizer, we recommend using 
dSpacer and the Chemical Phosphorylation Reagent of Glen Research, Sterling, VA. 
No changes of coupling conditions and deprotection are needed from the standard 
procedure of phosphoramidite chemistry-based oligonucleotide synthesis (see Note 3). 



Autosticky PCR 



145 



AS-PCR primer 



AS-PCR product 



AS-PCR product - vector 
ligation junction 



PCR with Tag 



T4 DNA ligase 



-GGATCCS- 
-CCTAG A- 



B 



PCR with PJUNztA 



T4 DNA ligase 



-GGATS- 
■ CCTAG- 



Fig. 1. The diagram of AS-PCR, shown in the case of primers designed for use with aBamHl 
site. 'S', tetrahydrofuran abasic site. The dashed lines represent continuation of the DNA. A., 
AS-PCR primer for use with Taq polymerase. B., AS-PCR primer for use with Pfu or Vent 
polymerase. 



3.3. Vector Preparation (see Notes 4 and 5) 

The cloning vector should be cut with the two restriction endonucleases (generat- 
ing noncompatible 5' overhangs), and the vector fragment isolated by preparative gel 
electrophoresis. 

It is recommended to perform the two restriction cleavages one after the other, not 
together at the same time, because of the monitoring of the reactions. If possible, use 
the optimal, manufacturer-provided, usually 10X concentrated buffer for each diges- 
tion, to avert the adverse effects of the decreased specificity of restriction endonucle- 
ases ("star activity"). Consult the manufacturer's catalog if there is a common buffer 
recommended for the desired double digestion, namely a buffer in which both 
enzymes work with high efficiency, without significant star activity. If no common 
buffer is available for the two restriction endonucleases, and at least one of the 
enzymes can be heat inactivated, start with that enzyme. Consult the product descrip- 
tion of enzymes to find out if they can be heat inactivated (see Note 4). 

1. Assemble the first restriction digestion in a 1.5-mL reaction tube: 

a. Vector DNA 5 ug 

b. 10X buffer for first digestion 5 u,L 

c. First restriction endonuclease 10-40 U (see Note 6) 

d. Sterile deionized water up to 50 u,L 

The restriction endonuclease should always be added last to the mixture. Mix compo- 
nents with a pipet, and incubate at the temperature optimum of the first restriction endo- 
nuclease for 1 h. Monitor the progress of the digestion by running a 3-u.L aliquot of the 
mixture next to uncut vector DNA on an agarose gel. If the digestion is only partial, add 
more enzyme (see Note 7), and prolong the incubation. 

2. If there is a common buffer in which both enzymes perform well without star activity, go 
to step 2a. Otherwise, go to step 2b. 



146 Gal and Kalman 

a. If the first digestion is complete, add 10-40 U of the second enzyme to the mixture, 
mix with a pipet, and incubate at the temperature optimum of the second enzyme for 
at least 1 h (see Notes 6 and 7). If possible, monitor the second cleavage by agarose 
gel electrophoresis (see Note 8). Proceed to step 4. 

b. If the first enzyme can be heat inactivated, heat inactivate the enzyme following 
the manufacturer's recommendations, then go to step 3, (see Note 9). Otherwise, extract 
the first digestion mixture with an equal volume of a 25:24:1 mixture of phe- 
nol:chloroform:isoamylalcohol, either by vortexing, or in the case of plasmids larger 
than 10 kb, by inverting or flicking the tube several times. Spin in a table-top centri- 
fuge at 16,000g for 5 min. Carefully transfer the upper (aqueous) phase into a new 
1.5-mL reaction tube with a pipet. Go to step 3. 

3. Precipitate DNA by adding 0.1 volume of 3 M sodium acetate, pH 5.2 and 2.5 volume of 
cold 100% ethanol to 1 volume of DNA solution. Mix thoroughly by inverting the tube 
several times, and incubate at -20°C for 30 min. Pellet DNA by centrifugation at 16,000g 
in a table-top centrifuge for 10 min, discard supernatant, and wash pellet with 1 mL cold 
70% ethanol. Air-dry pellet and dissolve it in a volume of sterile deionized water as deter- 
mined below, then add the further components of the second restriction mixture. 

a. 10X buffer for second enzyme 5 [xL 

b. Second restriction endonuclease 10-40 U 

c. Sterile deionized water up to 50 [xL 

Mix components with a pipet and incubate at the temperature optimum for the 
second enzyme for at least 1 h. If possible, monitor the second cleavage with agarose 
gel electrophoresis (see Note 8). Proceed to step 4. 

4. The double-cut vector fragment should be isolated by preparative gel electrophoresis. 

3.4. Amplification (see Note 10) 

1. Assemble the following reaction mixture in a thin-wall PCR tube on ice: 

a. 10X PCR buffer 10 (xL 

b. 25 mM Mg 2+ salt (see Note 11) 8 |xL 

c. 5 tiiM (each) dNTP 4 |xL 

d. Template DNA Plasmid: 1-100 ng 

Genomic DNA: 100-250 ng 

e. 10 fxM upstream AS-PCR primer 10 [xL 

f. 10 \iM downstream AS-PCR primer 10 (xL 

g. Thermophilic DNA Polymerase Taq, Pfu: 2.5 U 

Vent: 2 U 
h. Deionized, DNA-free water up to 100 |xL 

2. If the thermal cycler operates without a heated lid, overlay the reaction mixture with 70 [xL 
light mineral oil. 

3. The recommended PCR program is: 

a. Initial denaturation 94°C, 4 min (see Note 12) 

b. 20-25 cycles of: 94°C, 1 min 

50-55°C (see Subheading 3.1.), 1 min 

72°C: Taq, Vent: 1 min per each kilobase, at least 1 min; Pfu: 
2 min per each kilobase, at least 1 min 

c. Final extension 72°C, 5 min 



Autosticky PCR 147 

3.5. Analysis and Purification of the AS-PCR Product 

Run 3 \\L of the amplification mixture on an agarose gel next to a suitable molecu- 
lar weight marker. 

1 . If the product with the expected molecular weight is absent, or is hardly visible, the reac- 
tion conditions should be optimized. 

2. If only a single fragment with the expected length is apparently present, it is advised to 
purify the product from the amplifying polymerase and residual unused primers with the 
QIAquick PCR Purification Kit (QIAGEN), or a similar kit (see Note 13). Alternatively, 
preparative gel electrophoresis is also sufficient. 

3. If unexpected bands are also present, it is strongly recommended to purify the fragment 
of interest by preparative gel electrophoresis. 

3.6. Ligation 

1 . After purification, the concentrations of the AS-PCR product and the isolated, double-cut 
vector fragment should be estimated with agarose gel electrophoresis. An aliquot should 
be run next to a DNA standard of known concentration, e.g., a DN A size marker in which 
the amount of DNA running in a nearby band is known. 

2. Assemble the ligation reaction as follows: 

a. Vector DNA (double-cut, isolated) 50 ng 

b. AS-PCR product (in fivefold molar excess over the vector; see Note 14) 

c. 10X T4 DNA ligase buffer 1 .5 uL 

d. T4 DNA ligase 1.5 Weiss U 

(= 100 cohesive end ligation U) 

e. Sterile deionized water up to 15 uL 
Incubate overnight at 16 °C. 

3. Also assemble a control reaction in which the AS-PCR product is replaced with the buffer 
that the AS-PCR product is dissolved in. 

3.7. Transformation 

To elevate the efficiency of transformation, T4 DNA ligase might be inactivated 
before transformation by incubation at 65°C for 10 min. 

1. Thaw DH5ct E. coli competent cells on ice. 

2. Add 5 uLof the ligation mixture to 100 u,L of cells. Mix by flicking the tube a few times. 

3. Incubate cells on ice for 20 min. 

4. Heat shock cells at 42°C for 30 s. 

5. Place cells back on ice for 2 min. 

6. Add 900 u,L of LB broth, and incubate cells at 37°C for at least 30 min, but no longer 
than 60 min. 

7. Plate 100 and 900 uL of the transformation mixture onto LB/1.5% agar plates containing 
the appropriate antibiotic (see Note 15). Alternatively, plate 100 uL of the transformation 
mixture, and keep the remainder at 4 C C. Usually, transformed cells can be kept at 4°C for 
a day without substantial loss of viability. 

8. Incubate plates overnight at 37°C (see Note 16). 



148 Gal and Kalman 

3.8. Plasm id Purification 

1. Pick colonies with an inoculating loop or sterile toothpicks, and inoculate 3 mL LB broth 
containing the appropriate antibiotic (see Note 15). Shake overnight at 37°C. The bacte- 
rial culture should reach stationary phase. 

2. Pellet cells from 1.5 mL of the bacterial culture in a 1.5 mL reaction tube by spinning in 
a table-top centrifuge at 16,000g for 1 min. Discard supernatant. 

3. Repeat step 2 with the rest of the bacterial culture. 

4. Resuspend cells in 300 u,L Sol-1 with vortexing. 

5. Add 300 [xL Sol-2, and invert the tube several times. Do not vortex or shake the tube at 
this point. Incubate at room temperature for 5 min. 

6. Add 300 u.L ice-cold Sol-3, and invert the tube several times. Do not vortex or shake the 
tube at this point. Place on ice for 10 min. 

7. Pellet the precipitated material by spinning in a table-top centrifuge at 16,000g for 10 min. 

8. Carefully transfer supernatant (approx 850 uL) with a pipet into a new 1.5-mL reac- 
tion tube. 

9. Precipitate nucleic acids by adding 600 uL of room-temperature isopropanol. Mix thor- 
oughly by inverting the tube several times, and spin at 16,000g for 10 min in a table-top 
centrifuge. 

10. Discard supernatant. Wash pellet with 1 mL cold 70% ethanol. 

11. Air-dry pellet. 

12. Disolve pellet in 180 u,L of TE buffer. 

13. Add 20 uL 100 (xg/mL RNaseA solution. Incubate at 37°C for 1 h. 

14. Extract the RNaseA mixture with 200 uL phenol:chloroform:isoamylalcohol (25:24:1) by 
vortexing, or in the case of plasmids larger than 10 kb, by inverting the tube several times. 

15. Spin at 16,000g for 5 min in a table-top centrifuge. Transfer upper (aqueous) phase into a 
new 1.5-mL reaction tube. Add 20 uL 3 M sodium acetate, pH 5.2 and 500 uL cold 100% 
ethanol. Mix thoroughly by inverting the tube several times. 

16. Incubate at -20°C for 30 min. 

17. Spin at 16,000g for 10 min in a table-top centrifuge. Discard supernatant. Wash pellet 
with 1 mL cold 70% ethanol. Air-dry pellet. 

1 8. Dissolve pellet in TE buffer or sterile deionized water. (In the latter case, it is important to 
store the sample at -20°C for prolonged periods of time.) The recommended volumes: 

a. High copy number plasmids (e.g., pBluescript) 100 |xL 

b. Intermediate copy number plasmids (e.g., pBR322) 30 uL 

c. For low copy number plasmids (e.g., pSClOl), decrease the volume further. 

3.9. Analysis of Clones 

3 |iL of the plasmid solution should be checked by restriction digestion with 
enzymes cleaving in the flanking regions of the insert (see Note 17). In the case of the 
Taq AS-PCR products, if the primers were designed to regenerate the cloning sites, 
the resulting clones can be checked by digestion with the restriction endonucleases 
used for the cloning. Positive clones should also be sequenced. 

4. Notes 

1. For useful hints in the preparation of stock solutions, turn to ref. 19. 

2. Do not use tandem abasic sites in AS-PCR primers (11). Do not forget to phosphorylate 
the 5' termini of the primers (11). 



Autosticky PCR 149 

3. Detailed information on dilution and coupling conditions for various synthesizers is avail- 
able at http://www.glenres.com/. 

4. Detailed information on restriction digestion conditions, recommended buffers for double 
digests, heat inactivation of restriction endonucleases, etc. is available at http://www. 
neb.com/. 

5. For a detailed description of analytical and preparative agarose gel electrophoresis, turn 
to (19). For the recovery of the DNA from a cut-out gel slice, we recommend to use the 
QIAexII Gel Extraction Kit (QIAGEN) for its high yield, but several other methods are 
also available, e.g., phenol extraction of low melting temperature agarose gel (19), low- 
speed centrifugation (22), or electroelution (23). 

6. In most of the cases, 2 fxL of a manufacturer-provided restriction endonuclease stock, 
usually corresponding to 10-40 U of enzyme, is enough for the digestion of 5 u.g 
vector DNA. 

7. Restriction endonucleases are usually provided in a stock solution containing 50% glyc- 
erol. The final concentration of glycerol in the restriction mixture should not exceed 5%, 
otherwise it might elicit star activity of certain restriction endonucleases. According to 
the aforementioned, the volume of the enzyme added as a 50% glycerol stock should not 
exceed 10 % of the volume of the mixture. 

8. If the second cleavage does not remove at least 5% of the single-cut vector, e.g., if both 
cleavages occur within a polycloning site of a vector, monitoring of the second cleav- 
age by agarose gel electrophoresis might be difficult, or even impossible. In this case, 
do a separate, analytical scale digestion to make sure that the second enzyme does cut 
the vector. 

9. Many restriction endonucleases can be inactivated with a 20-min incubation at 65°C. In 
some cases, an 80°C incubation might be necessary. However, certain restriction endonu- 
cleases survive even an 80°C heat treatment. In the latter case, a phenol:chloroform:iso- 
amylalcohol extraction of the DNA is necessary. For the thermal stability of a given 
enzyme, consult the manufacturer's product description. 

10. The presented conditions work well with most amplifications. However, optimization of 
certain conditions, e.g., concentration of the Mg 2+ , annealing temperature, number of cycles, 
amount of enzyme, concentration of the primers, etc. might be necessary in certain cases. 

11. For Taq polymerase, MgCl 2 is recommended; for Pfu and Vent, use MgS0 4 . Add the 
Mg 2+ salt only if it is not contained within the 10X PCR buffer. 

12. If an antibody-based "hot start" polymerase is used, a longer initial denaturation, or addi- 
tional cycles might be necessary; follow the manufacturer's instructions. 

13. It is strongly recommended to purify the AS-PCR product from the amplifying poly- 
merase before ligation. Polymerase and dNTP carryover from the PCR mixture caused 
self-circularization of the vector because of filling-in of the vector-borne 5' restriction 
overhangs, even at 16°C (10). It is also recommended to purify the amplification product 
from AS-PCR primers. An AS-PCR primer, due to its structure, could also ligate to vec- 
tor-borne 5' overhangs, decreasing the amount of the vector available for cloning. For the 
purification of the AS-PCR product from PCR mixture components, we used the 
QIAquick PCR Purification Kit (QIAGEN), but other methods should also work. 

14. The highest cloning efficiency was obtained when AS-PCR products were cloned right 
after amplification and purification. 

15. The recommended final concentrations of some frequently used antibiotics: ampicillin, 
100 [xg/mL; chloramphenicol, 30 u.g/mL; Kanamycin, 50 u.g/mL; streptomycin, 25 u.g/mL; 
tetracycline, 12.5 (ig/mL. 



150 Gal and Kalman 

16. The plate with the vector control should contain a low number of colonies (background) 
as compared to the plate with the AS-PCR product-vector ligation. Otherwise, probably 
at least one of the restriction digestions of the vector was only partial, or some solution 
might have been contaminated with DNase, and removal of the vector-borne sticky ends 
might have led to recircularization of the vector by blunt-ligation. 

17. If the cloned DNA fragment is short (below 500 basepairs), it might be necessary to 
digest more plasmid to generate visible amount of the insert on agarose gel. 

References 

1. Mullis, K. B. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a poly- 
merase-catalyzed chain reaction. Meth. Enzymol. 155, 335-350. 

2. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., et al. 
(1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA poly- 
merase. Science 239, 487-491. 

3. Scharf, S. J., Horn, G. T., and Erlich, H. A. (1986) Direct cloning and sequence analysis 
of enzymatically amplified genomic sequences. Science 233, 1076-1078. 

4. Jung, V., Pestka, S. B., and Pestka, S. (1990) Efficient cloning of PCR generated DNA 
containing terminal restriction endonuclease recognition sites. Nucl. Acids Res. 18, 6156. 

5. Kaufman, D. L. and Evans, G. A. (1990) Restriction endonuclease cleavage at the termini 
of PCR products. Biotechniques 9, 304-306. 

6. Schaaper, R. M., Kunkel, T. A., and Loeb, L. A. (1983) Infidelity of DNA synthesis asso- 
ciated with bypass of apurinic sites. Proc, Natl. Acad. Sci. USA 80, 487-491. 

7. Takeshita, M., Chang, C.-N., Johnson, F., Will, S., and Grollman, A. P. (1987) 
Oligodeoxynucleotides containing synthetic abasic sites. J. Biol. Chem. 262, 10,171-10,179. 

8. Paabo, S., Irwin, D. M., and Wilson, A. C. (1990) DNA damage promotes jumping be- 
tween templates during enzymatic amplification. /. Biol. Chem. 265, 4718-4721. 

9. Greagg, M. A., Fogg, M. J., Panayotou, G., Evans, S. J., Connolly, B. A., and Pearl, L. H. 
(1999) A read-ahead function in archaeal DNA polymerases detects promutagenic tem- 
plate-strand uracil. Proc. Natl. Acad. Sci. USA 96, 9045-9050. 

10. Gal, J., Schnell, R., Szekeres, S., and Kalman, M. (1999) Directional cloning of native 
PCR products with preformed sticky ends (Autosticky PCR). Mol. Gen. Genet. 260, 
569-573. 

11. Gal, J., Schnell, R., and Kalman, M. (2000) Polymerase dependence of Autosticky poly- 
merase chain reaction. Analyt. Biochem. 282, 156-158. 

12. Clark, J. M. (1988) Novel non-templated nucleotide addition reactions catalyzed by pro- 
caryotic and eucaryotic DNA polymerases. Nucl. Acids Res. 16, 9677-9686. 

13. Hu, G. (1993) DNA polymerase-catalyzed addition of nontemplated extra nucleotides to 
the 3' end of a DNA fragment. DNA Cell Biol. 12, 763-770. 

14. Lundberg, K. S., Shoemaker, D. D., Adams, M. W. W., Short, J. M., Sorge, J. A., and 
Mathur, E. J. (1991) High-fidelity amplification using a thermostable DNA polymerase 
isolated from Pyrococcus furiosus. Gene 108, 1-6. 

15. Manila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991) Fidelity of DNA synthesis 
by the Thermococcus litoralis DNA polymerase - an extremely heat stable enzyme with 
proofreading activity. Nucl. Acids Res. 19, 4967-4973. 

16. Chien, A., Edgar, D. B., and Trela, J. M. (1976) Deoxyribonucleic acid polymerase from 
the extreme thermophile Thermus aquaticus. J . Bacteriol. 127, 1550-1557. 

17. Tindall, K. R. and Kunkel, T. A. (1988) Fidelity of DNA synthesis by the Thermus 
aquaticus DNA polymerase. Biochemistry 27, 6008-6013. 



Autosticky PCR 151 

18. Horn, T. and Urdea, M. S. (1986) A chemical 5 '-phosphorylation of oligodeoxyribonu- 
cleotides that can be monitored by trityl cation release. Tetrahedron Lett. 27, 4705-4708. 

19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory 
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

20. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. ./. Mol. 
Biol. 166, 557-580. 

21. Newton, C. R., Holland, D., Heptinstall, L. E., Hodgson, I., Edge, M. D., Markham, A. F., 
and McLean, M. J. (1993) The production of PCR products with 5' single-stranded tails 
using primers that incorporate novel phosphoramidite intermediates. Nucl. Acids Res. 21, 
1155-1162. 

22. Heery, D. M., Gannon, F., and Powell, R. (1990) A simple method for subcloning DNA 
fragments from gel slices. Trends Genet. 6, 173. 

23. Zhen, L. and Swank, R. T. (1993) A simple and high yield method for recovering DNA 
from agarose gels. Biotechniques 14, 894-898. 



17 



A Rapid and Simple Procedure for Direct Cloning 
of PCR Products into Baculoviruses 

Tamara S. Gritsun, Michael V. Mikhailov, and Ernest A. Gould 
1. Introduction 

Recombinant baculovirus expression systems are among the most commonly used 
for expressing foreign genes in eukaryotic cells (1,2). This eukaryotic expression sys- 
tem became very popular for many reasons, including 1. potentially high-protein 
expression levels, 2. ease and speed of genetic engineering, 3. ability to accommodate 
large DNA inserts, 4. protein processing similar to higher eukaryotic cells (e.g., mam- 
malian cells), and 5. ease of insect cell growth (3-5). Owing to their popularity, there 
have been considerable advances in the development of recombinant baculovirus sys- 
tems to extend their range of application to include medicinally important pharma- 
ceuticals (6), vaccine development (7,8) and rapid-action biological insecticides (9). 
As an expression system, the baculoviruses have been improved and modified to facil- 
itate easy laboratory manipulation and high-level protein expression (10-12). 

Baculoviruses are a diverse group of insect viruses. They have a double-stranded 
circular DNA genome about 130 kb in length. The most commonly used baculovirus 
for protein expression is Autographa califomica nuclear polyhedrosis virus 
(AcMNPV). Conventionally, the foreign gene is inserted into the baculovirus expres- 
sion vector through a bacterial transfer vector, which contains two "recombinant arms" 
(RA), i.e., long baculovirus sequences consisting of 300-3000 nucleotides. These RA 
provide classical homologous recombination between the transfer vector and the 
baculovirus during cotransfection (see Fig. 1A). In some cases this conventional 
approach is impossible because of incompatibility between the eukaryotic sequences 
and the bacterial systems used for their cloning. One method that avoids the bacterial 
cloning stage is based on the direct ligation of foreign DNA into a specific restriction 
site (see Fig. IB) within the baculovirus (13,14). Another approach uses ligation 
between the transfer vector and the foreign DNA, followed by cotransfection of the 
purified ligation complex (see Fig. 1C) and the baculovirus DNA (15). 



From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 



153 



154 Gritsun, Mikhailov, and Gould 



S-p^rr Vf*^^ 




Fig. 1. Different cloning strategies of a protein gene. The baculovirus genome is shown as 
the larger ellipse with authentic gene designed for substitution (stripe-shafted arrow) and 
recombination arms (RA, gray boxes). The transfer vector is shown as the smaller ellipse with 
the gene destined to be cloned into the baculovirus (thick black arrow) flanked by the RA. 
Crossed dashed lines show homologous recombination resulting in genetic exchange between 
genes. Sites of ligation are depicted as shadowed stars. (A) The gene Y is cloned into transfer 
vector. (B) Direct ligation of gene to baculovirus DNA genome. (C) The gene is ligated to 
transfer vector. (D) PCR product of gene contains the truncated RA that enable homologous 
recombination. 



Here we present a novel alternative approach, by introducing foreign genes into 
baculoviruses without an intermediate bacterial stage. The method, which is based on 
the principal of homologous recombination, utilizes polymerase chain reaction (PCR) 
primers that contain 50 nucleotides of RA representing the site where recombination 
is required (see Fig. ID). The method is simple, rapid, and reliable and avoids the use 
of cumbersome techniques associated with enzymatic treatment and DNA purification. 
The method could be used not only for expression of genes, but also for cloning pur- 
poses when bacterial cloning of DNA with "difficult" sequences appears impossible. 

This chapter does not provide the details for manipulation of baculoviruses and 
baculovirus DNA because these procedures have been described many times in spe- 
cialized manuals and books (see Note 1). Moreover, many commercial companies 
offer ready-to-use kits supplying the full range of required chemicals, purified DNA, 
and manuals. Our method for direct PCR cloning could help scientists who are already 
familiar with the baculovirus expression system. For those who are not, we recom- 
mend the comprehensive laboratory guides (1,2) describing the details of virus growth, 
plaque assay, and virus genomic DNA purification. 

1.1. Theory of the Method 

The key elements of our PCR cloning method are to define the locus on the 
baculovirus genome for insertion of the PCR product and then to design appropriate 
primers with RA that will enable homologous recombination between the PCR prod- 
uct and the baculovirus. Here we present the sequence of the baculovirus polyhedrin 
locus with an inserted |3-galactosidase gene (lacZ) [baculovirus Acrp23-/acZ (16)] 



Direct Cloning into Baculoviruses 155 

PcfrlKdilH ... ftJhhPilriii 1'J.Iil-Jm. 

pMluilir '"" l»nnin»lr(T tmim 

IfdniJT 



I* 
iillii | F J. In ill In iMUv 

Fig. 2. Strategy for direct cloning of GFP gene into baculovirus genome. Restriction enzyme 
Bsu361 was used to linearize baculovirus DNA (AcRP23-/acZ) inside the (3-galactosidase gene. 
Both upstream and downstream primers for PCR contained sequences of the baculovirus 
polyhedrin locus (black boxes) and GFP gene (white arrows). The viral DNA and GFPPCR 
DNA were mixed and cotransfected into insect cells. The resulting recombinant virus expressed 
the 238 amino acid GFP, replacing expression of the (3-galactosidase gene. 



that in our experiments was replaced by the gene for green fluorescent protein (GFP 
describes the protein and gfp describes the gene) (see Figs. 2 and 3). We have chosen 
this example to demonstrate the principle of primer design. The same principles can 
be applied to different protein genes and to a broad variety of baculovirus expression 
vectors with different loci and different promoters. 

Fig. 2 represents the schedule for the insertion of gfp in place of the lacZ of 
AcRP23-/acZ baculovirus (16). |3-galactosidase is an enzyme that converts the white 
chromogenic substrate X-gal to a blue product. Parent virus AcRP23-/acZ expressing 
-galactosidase, under the virus polyhedrin promoter, forms blue-coloured plaques in 
5/21 insect cells under agarose overlay containing X-gal. The recombinant baculovirus 
AcRP23-GFP acquires gfp and loses lacZ during the homologous recombination event 
and therefore produces white plaques in cell culture. 

The GFP belongs to a family of fluorescent proteins from the jellyfish Aequorea 
victoria and it is an important reporter molecule for monitoring gene expression and 
protein localisation in vivo and in situ. The fluorescence excitation and emission 
spectra of gfp are similar to those of fluorescein, and the conditions used to visualise 
this fluorophore are also suitable for GFP (17). 

Fig. 3A depicts the nucleotide and amino acid sequences comprising the polyhedrin 
locus, for expression of the lacZ. The locus commences with the polyhedrin promoter 
followed by the nucleotide A for transcription initiation. It also contains sequences, 
encoding the C-terminal 188 amino acids of the polyhedrin gene followed by 
sequences important for polyhedrin transcription termination. The C-terminal 188 
amino acids of the polyhedrin protein gene are redundant and resulted from genetic 
manipulation during the construction of blue-plaque baculovirus AcRP23-/acZ (16). 
The sequences highlighted in bold at each terminus were selected as RA and are 
included in the primers for the PCR. 



156 Gritsun, Mikhailov, and Gould 

A 1 

^Transcription initiation 
4449 -ATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAAC^GTTTTGTAATAAAAA 
4520 1 

AACCTATAAATCCGGATCTGAGCTTGGGATCTCTATAATCTCGCGCAACCTATTTTCCCCTCGAA 

w lacZ 
CACTTTTTAAGCCGTAGATAAACAGGCTGGGACACTTCACATGAGCGAAAAATACATCGTCACCT 

MSEKYIVTW 

GGGACATGTTGCAGATCCATGCACGTAAACTCGCAAGCCGACTGATGCCT ► 

DMLQIHARKLASRLMP 

lacZ stop codon | SV40 terminator JC-terminus 188 amino acids of polyhedrin gene 



I SV40 terminator JC 



► 5211 -AAGGAGTTTGCACCAGACGCACCTCTGTTCACTGGTCCGGCGTAT 

, KEFAPDAPLFTGPAY 

y 5256 (polyhedrin stop codon) 

TAAAACACGATACATTGTTATTAGTACATTTATTAAGCGCTAGATTCTGTGCGTTGTTGA p. 

transcription termination 
► 5627 -TTATGCGCTTTTGTATTTCT-5 659 ► 



" T Transcription initiation 

4449 - ATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAA 
4520^ gfp 

AACCTATAAATATGGCTAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTT ► 

MASKGEELFT, GVVPIL 
stop codon y 5256 
►ATTACACATGGCATGGATGAACTATACAAATAAAACACGATACATTGTTATTAGT 

ITHGMDELYK* 



ACATTTATTAAGCGCTAGATTCTGTGCGTTGTTGA 
transcription termination 



► 5627 -TTATGCGCTTTTGTATTTCT-5 659 

Fig. 3. Sequences of polyhedrin locus with cloned A lacZ and B gfp. Numbers correspond to 
the numeration of baculovirus AcMNPV (Accession number NC001623). Solid arrows indi- 
cate the main functional features of the locus — transcription initiation and stop codon of lacZ, 
SV40 terminator of lacZ transcription, the redundant sequences of C-terminal 188 amino acids 
of polyhedrin gene, including stop codon in position 5256 and the transcription termination 
signal of the authentic polyhedrin gene. Dashed arrowhead lines correspond to sequences not 
included in this picture. Translation of the beginning of the lacZ and gfp and the end of the 
polyhedrin gene is shown. The sequences highlighted in bold were used to design primers for 
cloning the gfp in this locus. 



As the result of insertion of the gfp into the locus instead of the lacZ (Fig. 3B), the 
pretranslation region preceding the first initiation codon is shortened and transcription 
termination is provided by the transcription termination signal following the stop 
codon of the polyhedrin gene, not by the SV40 terminator as required for transcription 



Direct Cloning into Baculoviruses 157 

termination of lacZ. The sequences highlighted in bold from each terminus represent 
the final version of the upstream and downstream primers for the PCR including 50 
nucleotides of baculovirus polyhedrin locus sequence followed by either 19 or 21 
nucleotides from the 5' or 3' ends, respectively, of the gfp (see Notes 3 and 4). 

Therefore the following principles to design primer sequences with RA must be 
established. 

1 . RA for the upstream primers should include the 5' untranslated region and initiation codon 
ATG. The RA should follow the promoter. The appropriate 5' untranslated regions pro- 
vide high levels of protein expression. The best way to select the 5' untranslated region is 
to use the sequence that was already used for the expression of the authentic gene to be 
substituted. As illustrated earlier, in some cases the 5' untranslated region can be short- 
ened without a significant reduction in protein expression. Each particular case has to be 
considered carefully. 

2. RA for the downstream primer should be placed behind the stop codon and should 
include it. The sequences of the termination transcription signal must follow the RA. If 
the transcription termination signal is not known, it is safe to place the RA immediately 
after the stop codon of the authentic gene. 

3. The length of "protein" sequence in the "recombinant" primers depends only on the tem- 
perature of primer annealing to template during PCR. For example, if the PCR product is 
less than 2 kb and nonspecific DNA products are not expected, the temperature of anneal- 
ing could be in the range 45-60°C with the length of the "protein" part of the primers 
about 16-20 nucleotides. If the expected PCR product is longer than 2 kb and amplifica- 
tion of nonspecific PCR products is expected, then the calculated annealing temperature 
of the "protein" part of the primers must be 72-80°C with a length of 24-30 nucleotides 
(18-20). 

4. In our experience, 50 nucleotides of RA are sufficient for recombination. Among the 84% 
of white plaques formed by the recombinant virus about 20% produced green fluores- 
cence. We also tried 30 nucleotides of RA for the upstream primer only and found the 
same rate of recombination. We did not try shorter RA, but it is quite possible that shorter 
RA would work albeit with lower recombination efficiency. 

2. Materials 

2.1. Buffers 

1. TE buffer: 10 mM Tris-HCl, pH 8.0, 2 rnM ethylene diaminetetraacetic acid (EDTA). 

2. 10XTBE buffer for (1 L): 54 g Tris-base, 27.5 g boric acid, 4.65 g EDTA (21). 

2.2. Chemicals and Equipment for PCR 

1. Mixture of deoxynucleotide 5-triphosphates (dNTPs) (Amersham): 10 mM each. 

2. Liquid paraffin (Fisons Scientific Equipment, Inc. Griffin & George). 

3. pGFP (plasmid containing gfp) (CLONTECH Laboratories, Inc.)(see Note 4). 

4. Thermostable DNA polymerase (Taq) (Sigma-Aldrich) provided with appropriate 10X 
buffer (see Note 5). 

5. Agarose (electrophoresis grade) (Gibco-BRL). 

6. PCR equipment (thermal cycler) of any design. 

7. Tank, tray, and power pack of any design for the electrophoresis of DNA in agarose gel. 

8. Gel Extraction Kit (Qiagen). 



158 Gritsun, Mikhailov, and Gould 

2.3. Primers for the PCR (see Notes 3 and 4) 

1. GFP-BAC-up (baculovirus sequence is emboldened, initiation codon is in small letters): 
5TATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATatg 
GCTAGCAAAGGAGAAGAAC3'. 

2. GFP-BAC-down (baculovirus sequence is emboldened, stop codon is in small letters): 
5GCACAGAATCTAGCGCTTAATAAATGTACTAATAACAATGTATCGTGTTtta 
TTTGT ATAGTTC ATCC ATG3 ' . 

2.4. Cells 

All types of work associated with cells must be carried out aseptically using sterile 
plasticware and reagents. 

Insect cells (5/21) should be grown in suspension using TCI 00 medium (Gibco-BRL) 
with 10% foetal calf serum (FCS) (Gibco-BRL) (see Note 1) in 500 mL spinner 
bottles with a magnetic stirrer. For routine culture, cells should be grown to a density 
of 1 x 10 6 /mL and then subcultured to another bottles at a dilution rate 1:3-1:4 usually 
twice per wk. 

2.5. Chemicals and Equipment for Baculovirus Transfection 
and Plaque Assay 

1. Purified DNA of baculovirus AcRP23-/arZ reconstituted in TE buffer at a concentration 
of 500 ng/uL (see Note 1). 

2. Restriction endonuclease Bsu36 I (New England Biolabs Ltd.) provided with appropriate 
1 OX buffer. 

3. Fluorescence microscope. 

4. Medium TC100 (Gibco-BRL) with 10% FCS (Gibco-BRL). 

5. Lipofectin™ (Gibco-BRL). 

6. Low melting agarose (Flowgen). Prepare 3% agarose in water using 100 mL glass bottles, 
sterilize it by autoclaving and keep at room temperature until use. 

7. 24-well sterile plastic plates for cell culture (NUNC™). 

8. 33-mm sterile plastic dishes for cell culture (CORNING™). 

9. 96-well sterile plastic plates for cell culture (NUNC™). 

10. Sterile glass Pasteur pipet. 

11. Rubber teat. 

12. Stain: sterile stock neutral red 3.3 g/mL (SIGMA). 

13. Chromogenic substrate for p-galactosidase X-gal (Melford Laboratories Ltd.). Prepare 
2% stock using dimethylformamide (BDH) and store at -20 C C. 

3. Methods 

3.1. PCR Amplification of Protein Gene 

1. Amplify gfp gene in PCR (see Note 5): 
1 OX buffer 10 uL 

dNTPs(lOmM) 2 uL 

Primer GFP-BAC-up (10 pM) 5 uL 

Primer GFP-BAC-down (10 pM) 5 uL 



Direct Cloning into Baculoviruses 159 



1].22 




im: 



U.UB 
hi S3 







<< ] 1 if fih 



I 



Fig. 4. Analysis of PCR product of gfp by electrophoresis in 1% Agarose gel. Molecular 
weight marker DNA (in kb) is shown on the left. The arrow indicates the positions of gfp 
PCR DNA. 



Water 76.5 uL 

pGFPDNA(10ng/(L) 1 ixL 

Taq polymerase (5 U/(L) 0.5 fxL 

2. Use the following PCR program: 3 cycles at 95°C for 40 s, 52°C for 1 min and 72°C for 
1 min, followed by 27 cycles at 95°C for 40 s and 72°C for 1 min (see Note 6). 

3. Test 5 u.L of PCR product in 1% agarose gel using TBE buffer. The derived PCR prod- 
uct in our case consisted of 860 base pairs (see Fig. 4). The 50 nucleotides of RA did 
not interfere with PCR (see Note 3). The additional DNA band represents nonspecific 
priming. 

4. Repeat PCR (if successful in the first place) in 3-5 tubes and pool the PCR products to 
obtain 10-50 mg of DNA. 

5. Separate the amplified DNA from nonspecific DNA by electrophoresis of the pooled 
PCR product in 1% agarose gel (see Note 7). 

6. Extract DNA from the excised agarose using Qiagen Gel Extraction Kit and adjust con- 
centration to 500 ng/(.iL. 



160 Gritsun, Mikhailov, and Gould 

3.2. Transfection Experiments 

All following procedures should be carried out aseptically and must be performed 
in a cabinet that provides HEPA-filtered air. 

1. Linearize baculovirus AcRP23-/ac\Z DNA (12) (see Notes 1 and 2) using a unique restric- 
tion site (Bsu361 in our case) within the lacZ. The linearization of circular DNA signifi- 
cantly reduces the appearance of unwanted parent blue-plaque viruses after transfection. 
Carry out the following reaction in a volume of 100 uL: 

DNA (500 ng/uL) 10 uL 

1 OX buffer 10 uL 

Water 80 uL 

Bsu36l (10 U/uL) 2 uL 

After incubation at 37°C for 2 h add the additional 2 uL of Bsu36l and incubate the 
mixture for another 2 h. 

2. Seed dishes (35 mm) with 1-1.5 x 10 6 5/21 cells and incubate at 28°C for 2 h to allow 
cells to attach to the plastic. 

3. Prepare the following mixture: 

Linearized baculovirus DNA (from Step 1) (see Notes 1 and 2) 1 uL 

PCR product (Step 6) 1 uL 

Water 48 uL 

4. Dilute the commercial Lipofectin™ 1:10 with water in a volume of 50 uL and add it 
dropwise to the mixture of baculovirus DNA and PCR product (Step 3). Incubate the 
mixture for 20 min at room temperature. 

5. Wash the cells on 35 mm dishes (Step 2) with 1 mL TC100 medium without serum and 
add 1 mL of TCI 00 medium without serum. 

6. Add the mixture of Lipofectin and DNA from Step 4 dropwise to the dishes, gently swirl- 
ing, and incubate for 5-20 h (as convenient) at 28°C. 

7. Remove supernatant medium from cells, add 1 mL of TC100 medium with 10% FCS and 
incubate at 28°C for 3 d. 

8. Harvest the supernatant medium (recombinant virus stock) in a sterile container and keep 
it at 4°C, before analysis for virus by plaque assay. 

3.3. Plaque Assay 

1. Seed 24 35-mm dishes with 1-1.5 x 10 6 5/21 cells in 2 mL of TC100 medium containing 
10% FCS and incubate at 28°C for 2 h to allow cells to attach. 

2. Make 10X dilutions of supernatant medium from transfection experiments (Subheading 
3.2., step 8) as follows: place 90 uL of TC 100 medium in 6 wells of a 96- well microtitre 
plate or suitable sterile vials; Add 10 uL of recombinant virus stock (Subheading 3.2., 
step 8) to the first well and then discard it; do not use the same tip to mix up liquid in 
the first well. Use a second tip to pipet the mixture into the first well, then using the same 
second tip transfer 10 uL from the first virus dilution to the second well and repeat the 
procedure until a final virus dilution of 1 x 10~ 6 is reached. 

3. Aspirate the medium from the 35-mm dishes from Step 1 and quickly apply four replicate 
aliquots of 0.1 mL of each dilution from supernatant medium (Step 2) starting with 
the 1 x 10" 6 dilution. Try to place the diluted virus stock in the center of the dish and 
make sure that the liquid is spread thoroughly across the cell monolayers. Using the same 
pipet, continue to apply the next higher concentration of virus to appropriate dishes. To 
avoid the risk of the monolayer drying out during this process, remove the medium from 



Direct Cloning into Baculoviruses 161 

four dishes and apply the inoculum, then prepare four more dishes, etc. Incubate for 1 h at 
room temperature with gentle agitation every 15 min. Make sure that the monolayers are 
covered completely with the liquid all the time. 

4. During this 1-h period of virus adsorption prepare the agarose overlay: 

a. Melt 3% agarose stock in a microwave oven and cool in a water bath at 42°C. 

b. Prewarm TC100 medium containing 10% FCS in water bath at 37 C C. 

5. Aspirate 0.1 mL of virus inoculum from 35 mm dishes from Subheading 3.2., step 3. 

6. Mix melted 3% agarose with equal volume of prewarmed TC100 medium containing 
10% FCS and gently apply 2 mL to the center of the 35 mm dish from Step 5. Let the 
agarose solidify for 15-20 min, add 1 mL TCI 00 medium containing 10% fetal calf serum 
(FCS) and place dishes in incubator at 28°C. 

7. The plaques will have developed by day 3 or 4. To visualize plaques, gently remove 1 mL 
of liquid covering the agarose overlay in each dish and replace it with 1 mL of TCI 00 
medium (without serum) containing 15 uL of 2% X-gal solution and 50 uL of neutral red 
solution (3.3 g/mL). Allow cells to stain for 16 h at 28°C. The parent virus will form blue 
plaques while recombinant virus will form white plaques on the red background. 

3.4. Evidence of Protein Expression (see Note 7) 

1 . To reveal expression of GFP, seed 24-well culture plates with 2-3 x 10 5 S/21 cells in 1 mL 
of TC100 medium containing 10% FCS and incubate at 28°C for 2 h to allow the cells to 
attach (the cells should form semiconfluent monolayers). 

2. Remove the stain covering the agarose from dishes described in Subheading 3.3., step 7, 
leave the dishes open in the hood to dry, a little, for 30 min. 

3. Using a wetted glass Pasteur pipet fitted with a rubber teat, stab the agarose immediately 
over a single well-defined white plaque. Try to pick-up the piece of agarose and cells that 
immediately cover a single plaque and then inoculate this directly into the medium above 
a cell monolayer of one well in a 24-well plate (from Step 1). Rinse the pipet 2-3 times 
with the medium in this well. Using a fresh sterile pipet each time, repeat this process for 
a large number of white plaques. 

4. Incubate the plates at 28°C for 3-4 d. 

5. To observe green fluorescence produced by recombinant baculoviruses remove nearly all 
the medium from 24-well plates (leave a small amount of medium to prevent drying of 
cells) and site the ultraviolet (UV) objective of the UV-microscope (with a filter for 
fluorescein) directly over each well. Look for fluorescence in the cells of the monolayer 
(see Note 8). A bright green color in the cells of the monolayer indicates that the recom- 
binant baculovirus is present. 

4. Notes 

1 . Different commercial companies (CLONTECH, Gibco-BRL, NOVAGEN,) offer a wide 
variety of baculovirus vectors and kits with purified DNA and provide comprehensive 
guidance. The purified and linearized DNA from kits might also be used as a cloning 
vector for PCR products of different genes. The choice of baculovirus vector is frequently 
defined by the screening system for the recombinant viruses and depends on the selective 
marker/reporter gene inserted in the baculovirus vector. 

2. The choice of baculovirus vector and locus for the expression depends on the nature of 
the intended cloning procedure. The reporter proteins could be cloned in a baculovirus 
vector even without preliminary linearization. Previously we described a cloning proce- 
dure for the (3-glucuronidase gene in another locus (P10) of PacBac6 baculovirus without 



162 Gritsun, Mikhailov, and Gould 

preliminary linearization of circular DNA (22). The resulting stock of recombinant viruses 
was heavily contaminated with parent virus forming white plaques in cell culture in the 
presence of chromogenic substrate X-(3-D glc. Nevertheless, a single blue plaque 
expressing (5-glucuronidase was easily visualized. 

3. In our experience the precise length of the primers is not critically important for success- 
ful PCR. The presence of long 50 nucleotide "tails" representing the RA of baculoviruses 
does not interfere with amplification of even longer template (up to 10 kb) in PCR 
(unpublished results). 

4. The sequences at either end of gfp vary depending on the sources of the information 
(accession numbers U19276, U36201). 

5. The experimental protocols presented here for the PCR are suitable for the amplification 
of short templates. For the amplification of long PCR templates the conditions may be 
different (18-20). Many companies (Perkin Elmer, Promega, Advanced biotechnology, 
Bioline) offer extension PCR amplification kits that supply chemicals and manual guides 
for the production of long PCR molecules. 

6. The programme for long PCR is different from those described above; it includes 72°C 
for annealing and a longer time for the extension depending on the length of the amplified 
region (18-20). 

7. Confirmation of expression of proteins other than fluorescent proteins can be obtained by 
more conventional methods, for example, by analysis of infected cell lysates from each 
well of 24-well plates, by protein electrophoresis in polyacrylamide gels and/or 
immunoblotting. If the expression of protein is not required and the baculovirus vector is 
used only for cloning purposes, the evidence of successful cloning can be produced by 
extracting the DNA from the supernatant medium of 24-well plates as described else- 
where (1,2) and using it as a template for a PCR test. 

8. We tested 48 white plaques and found 10 positive for green fluorescence, i.e., about 20% 
(22). For comparison, the use of a transfer vector containing genes designed for cloning 
in baculoviruses routinely gives 80-90% recombinant viruses (1,2). Nevertheless, when 
we tested recombinant virus DNA for the presence of cloned PCR product (by PCR) we 
found quite a high level of cloning (more than 90%) (unpublished). The reason for the 
lower efficiency of protein expression is not clear, but it does not detract from the useful- 
ness of the principle. Indeed this method has been used by us when bacterial cloning has 
proved to be virtually impossible. 

References 

1. King, L. A. and Possee, R. D. (1992) The Baculovirus Expression System: A Laboratory 
Guide, Chapman and Hall, London, U.K. 

2. O'Reilly, D. R., Miller, L. K. and A., L. V. (1994) Baculovirus Expression Vectors: A 
Laboratory Manual, Oxford University Press, New York. 

3. Possee, R. D., Thomas, C. J., and King, L. A. (1999) The use of baculovirus vectors for the 
production of membrane proteins in insect cells. Biochem. Soc. Trans. 27, 928-932. 

4. Murhammer, D. W. (1991) Review and patents and literature. The use of insect cell cul- 
tures for recombinant protein synthesis: Engineering aspects. Appl. Biochem. Biotechnol. 
31,283-310. 

5. Kidd, I. M. and Emery, V. C. (1993) The use of baculoviruses as expression vectors. Appl. 
Biochem. Biotechnol. 42, 137-159. 

6. Kutchan, T. M. (1996) Heterologous expression of alkaloid biosynthetic genes — a review. 
Gene 179,73-81. 



Direct Cloning into Baculoviruses 163 

1 . van Oirschot, J. T. (1999) Diva vaccines that reduce virus transmission. ./. Biotechnol. 73, 
195-205. 

8. Kelly, E. P., Greene, J. J., King, A. D., and Innis, B. L. (2000) Purified dengue 2 virus 
envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice. 
Vaccine 18, 2549-2559. 

9. Bonning, B. C. and Hammock, B. D. (1996) Development of recombinant baculoviruses 
for insect control. Annu. Rev. Entomol. 41, 191-210. 

10. Belyaev, A. S., Hails, R. S., and Roy, P. (1995) High-level expression of five foreign 
genes by a single recombinant baculovirus. Gene 156, 229-233. 

11. Kitts, P. A. and Possee, R. D. (1993) A method for producing recombinant baculovirus 
expression vectors at high frequency. Biotechniques 14, 810-817. 

12. Kitts, P. A., Ayres, M. D., and Possee, R. D. (1990) Linearization of baculovirus DNA 
enhances the recovery of recombinant virus expression vectors. Nucl. Acids Res. 18, 
5667-5672. 

13. Ernst, W. J., Grabherr, R. M., and Katinger, H. W. (1994) Direct cloning into the 
Autographa californica nuclear polyhedrosis virus for generation of recombinant 
baculoviruses. Nucl. Acids Res. 22, 2855-2856. 

14. Lu, A. and Miller, L. K. (1996) Generation of recombinant baculoviruses by direct clon- 
ing. Biotechniques 21, 63-68. 

15. Khromykh, A. A., Meka, H., and Westaway, E. G. (1995) Preparation of recombinant 
baculovirus by transfection of a ligated cDNA fragment without prior plasmid amplifica- 
tion in E. coli. Biotechniques 19, 356-360. 

16. Mann, S. G. and King, L. A. (1989) Efficient transfection of insect cells with baculovirus 
DNA using electroporation. ./. Gen. Virol. 70, 3501-3505. 

17. Kain, S. R., Adams, M., Kondepudi, A., Yang, T. T., Ward, W. W., and Kitts, P. (1995) 
Green fluorescent protein as a reporter of gene expression and protein localization. 
Biotechniques 19, 650-655. 

18. Gritsun, T. S. and Gould, E. A. (1995) Infectious transcripts of tick-borne encephalitis 
virus, generated in days by RT-PCR. Virology 214, 61 1-618. 

19. Gritsun, T. S. and Gould, E. A. (1998) Development and analysis of a tick-borne encepha- 
litis virus infectious clone using a novel and rapid strategy. ./. Virol. Meth. 76, 109-120. 

20. Cheng, S. and Kolmodin, L. (1997) XL PCR amplification of long targets from genomic 
DNA, in PCR Cloning Protocols:From Molecular Cloning to Genetic Engineering, 
Vol. 67 (White, B. A., ed.), Humana, Totowa, pp. 17-29. 

21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory 
Manual, Vol. 3 (Nolan, C., ed.), Cold Spring Harbor Laboratory Press, New York, pp. B23. 

22. Gritsun, T. S., Mikhailov, M. V., Roy, P., and Gould, E. A. (1997) A new, rapid and 
simple procedure for direct cloning of PCR products into baculoviruses. Nucl. Acids Res. 
25, 1864-1865. 



Ill 

Mutagenesis and Recombination 



18 

PCR Approaches to DNA Mutagenesis 
and Recombination 

An Overview 
Binzhang Shen 

1. Introduction 

Current polymerase chain reaction (PCR) innovations provide powerful tools for 
the cloning of previously unknown genes as well as characterization of their functions. 
Examples of the latter used include PCR-mediated in vitro mutagenesis and recombi- 
nation of the cloned genes. PCR approaches have become the method of choice to 
generate arrays of predefined mutations or recombination within the gene of interest. 
In the postgenomic era, these mutants or recombinants are highly desirable for the study 
of functional genomics, gene expression, protein structure-function relationships, pro- 
tein-protein interactions, protein engineering, and in vitro evolution of enzymes. 

There are numerous PCR-based approaches to DNA mutagenesis and recombina- 
tion; therefore, a complete coverage in this review of methods and new developments 
for such a rapidly advancing field is impractical. Instead, a selected subset of basic and 
representative approaches will be presented. These approaches are the prototypes upon 
which many of the other designs in the current literature are based. Interested readers 
should also refer to a previous review (1). 

PCR-mediated nucleotide changes, deletions, or insertions can be accomplished by 
performing PCR synthesis reactions with carefully chosen or modified PCR reaction 
components. One strategy for this kind of PCR mutagenesis is based on the principle 
of "misprinting" (2). Because mismatches between templates and primers are toler- 
ated under certain PCR conditions, primers can be designed to include predefined 
changes (so-called mutagenic primers). Other strategies make use of either the built-in 
high error rate of Taq DNA polymerase or degenerate base analogs such as deoxy- 
inosine triphosphate (dl) in the PCR reaction. In general, mutagenic primer PCR 
approaches are used for introducing site-directed mutagenesis (SDM) into the genes 
of interest, whereas Taq DNA polymerase or base analog PCR approaches are useful 
in creating random and extensive mutagenesis (REM) in the target gene. 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

167 



168 Shen 

A novel strategy to make recombinant molecules is to use PCR-mediated in vitro 
recombination. It involves formation of a heteroduplex intermediate between the two 
fragments to be recombined. There are two basic approaches to this PCR mediated in 
vitro recombination. In the first approach, PCR is employed to generate two overlap- 
ping primer-tailed genes capable of forming heteroduplex intermediates. Extension of 
the heteroduplex generates the construct of interest. In the second approach, new het- 
eroduplex intermediates are formed and extended following each PCR cycle until the 
chimeric constructs become full-length. Approaches have also been developed which 
utilize the in vivo homologous recombination strategy to create recombinants from 
transformed PCR products. 

In practice, which PCR strategy or approach to choose depends on the researcher's 
objective (SDM, REM or recombination), the template used (linear or circular), and 
the efficiency desired. In some instances, modification of the basic approaches is nec- 
essary, whereas in others, several approaches could be combined to achieve maximum 
performance. A comprehensive comparison of the basic PCR approaches discussed in 
the present review is summarized in Table 1 and Fig. 1. 

2. Mutagenic Primer 

A perfect base match between templates and primers is favored under most PCR 
conditions. Mutagenic primers or mismatched primers, albeit less efficiently, can direct 
DNA polymerization under conditions where 

1 . A perfect match is not present; 

2. Lower annealing temperature is applied; 

3. A mismatched nucleotide is located at the 5' end or in the middle of a long mutagenic primer. 

A mutagenic primer can be designed to include nucleotide substitutions, small dele- 
tions or insertions into its nucleotide sequence. As primers are incorporated into the 
final PCR products, so are these changes. In general, mutagenic primers are used either 
to create mutations in the target gene or to facilitate e cloning of the PCR products. 

3. Location of Mutations 

The mutagenic primer also defines the location of mutations. After a simple 
mutagenic PCR (SPCR) (3, Table 1-1.1 and Fig. 1-1.1) using a mutagenic primer, 
mutations are introduced into the PCR products. Mutations generated in this manner 
are usually restricted to the termini of the final PCR products. 

To overcome this limitation of SPCR, Ho et al. (4) devised overlap extension PCR 
(OEPCR). In OEPCR (3, Table 1-1.2 and Fig. 1-1.2), two overlapping primers and 
two flanking primers are used. Two separate SPCR amplifications are done, with each 
using one overlapping primer and one flanking primer. The two PCR products are then 
annealed and extended. The resulting full-length DNA is either used directly or further 
PCR amplified with the two flanking primers. The use and design of two overlapping 
primers is key to OEPCR. These primers, when mutagenic, allow the introduction of 
mutations throughout the whole construct. One drawback of OEPCR is its low effi- 
ciency, which is primarily because of short sequence homology between the overlap- 
ping primers. 



Table 1 

Major Advantages and Limitation of Different PCR Approaches for DNA Mutagenesis and Recombination 



Types of PCR 



Advantages 



Limitations 



1 . Basic mutagenic PCR 

1.1 Simple mutagenic PCR 

1.2 Overlap extension PCR 

1.3 Megaprimer PCR 

1.4 Inverse PCR 



simple and useful 

mutations anywhere in the PCR product, 

highly (100%) efficient, mutations anywhere 
desired 

for previously unknown genes 



mutations at the end of the PCR 
product, 50% mutants 

Low efficiency 

non-specific, non-template addition of 
bases at 3' end 

low efficiency 



<3> 



2. Restriction site PCR 

2.1 Modified restriction site PCR 

2.2 New restriction site PCR 



highly efficient 
highly efficient 



for previously cloned gene 
mutation sites restricted 



3.1 Marker couples PCR 



simple process 



low efficiency 



4. PCR-mediated recombination 

4.1 Gene SOEing 

4.2 DNA shuffling and Staggered 
Extension Process (STep) 

4.3 Recombinant circular PCR 

4.4 Recombinant PCR 



highly efficient (100%) 

random and extensive recombination 

good overall efficiency 
no post-PCR processing 



costly primers 

highly specific, efficient selection 
systems needed 

more PCR reactions 

overall efficiency 50% 



5. Nonmutagenic primer PCR 

5.1 Erroneous PCR 

5.2 Degenerate base analogs mediated PCR 

5.3 Stepwise elongation of sequence PCR 



Random and extensive mutations 



random and extensive mutations 



Extensive and specific mutations 



sequence-dependent error rate, 
biased mutations 

highly specific, efficient selection 
systems needed, 

multiple primers and PCR 



170 



Shen 



.A. 



/./ Simple mutagenic PCR(SPCR): primer 1 
is the mutagenic primer, 25-40 cycles, PCR 
products purified and ligated to vectors 



1- 



2^W 



1.3 Megaprimer PCR (MPPCR): PCR (2-3) 
product purified, used as a megaprimer, 
second PCR (1 -megaprimer) 



1- 






*+\r 3 



2.1 Modified restriction site PCR(MRPCR): 
polylinkers with restriction site at template 
ends, two PCR (1-3 and 2-4) combined, PCR 
(1-4), digested and ligated to vectors 



3.1 Marker coupled PCR (MCPCR) .marker 
gene activation primer 1 and target gene 
mutagenic primer 2, PCR (1-2), as a 
megaprimer, extended and transformed 




4.2 DNA shuffling and Staggered extension 
Process (StEP): various alleles mixed, 
fragmented by DNase I, random and briefly 
primed by repeated PCR cycles 

XXX X ^ „ 

5. / Erroneous PCR (EPCR): excess Taq 
DNA polymerase, high Mg++, Mn++, biased 
dNTP ratio 



5.2 Degenerate base analogs mediated 
PCR (DBAPCR): dITP in PCR reaction, 
dl integrated products as templates for 
further PCR 



1.2 Overlap extension PCR (OEPCR): two PCR 
(primers 1-2 and 3-4), combined, melted, 
annealed, extended and final PCR (primers 1-4) 




1.4 Inverse PCR (IPCR): end-to-end primers, 
PCR products re-circulated and transformed 



^^ 



^.2 



2.2 New restriction site PCR(NRPCR) .new 
restriction site and mutation on internal 
primer 2 and 3, two PCR (1-2 and 3-4), 
digested and re-ligated 

^^ :^= 

2* <— 4 

4. 1 Gene SOEing: linkers on primer 2 and 3, 
two PCR (1-2 and 3-4), overlap extension of 
1-2 and 3-4, finally PCR (1-4) 



2 4 

4. 3 Recombinant circular PCR (RCPCR): 
overlapping mutagenic primer land 4, two 
PCR (1-2 and 3-4), combined, annealed and 
transformed 




4.4 Recombinant PCR (RPCR): overlapping 
mutagenic primers 1 and 2, PCR (1-2) 
combined, annealed, transformed, in vivo 
recombination 




5.3 Stepwise elongation of sequence PCR 
(SESPCR): overlapping synthetic primers 
spans the known gene sequence, serial PCR 



Fig. 1. Design of different PCR approaches to DNA mutagenesis and recombination. Note: 
O inactive restriction site, • restriction site, □ inactive form, ■ active form, -A — p. muta- 
genic primer. 



Mutagenesis and Recombination Overview 171 

Megaprimer PCR (MPCR) (5) has been developed to improve the efficiency of 
OEPCR. Instead of using a pair of overlapping primers, MPCR (3, Table 1-1.3 and 
Fig. 1-1.3) starts with a SPCR using an internal mutagenic primer and a flanking 
primer. PCR products generated in this manner are purified and used as a primer (hence 
"megaprimer") for the second round of PCR along with another flanking primer. Wild- 
type sequences are used as the template in both PCR reactions. Compared to OEPCR, 
MPCR uses fewer primers and a longer homology match between megaprimer and 
template, and this leads to a higher efficiency. A major limitation of MPCR is the 
nonspecific, nontemplate addition of nucleotides at 3' end of the megaprimer. 

Both OEPCR and MPCR approaches discussed above have an advantage over 
SPCR for generating mutations throughout the whole construct, but they also have 
limitations. To overcome the SPCR limitation, a circular template can be used. The 
gene of interest can first be cloned into a vector and amplified using two end-to-end 
primers by an inverted PCR (IPCR) approach (6, Table 1-1.4 and Fig. 1-1.4). The 
IPCR products are recirculated and amplified in Escherichia coli (E.coli). Depending 
on the location of the mutagenic primer-matched sequence within the template, muta- 
tions can also be introduced throughout the whole gene. 

4. Cloning Efficiency 

The low efficiency of ligation in the post-PCR cloning processing is a common 
problem experienced by most users of the above-mentioned approaches. Depending 
on whether a linear template or a circular template is available, either in vitro or in 
vivo recombination strategy could be used to improve post-PCR cloning efficiency. 
When linear templates have to be used, in vitro ligations of the PCR products are often 
necessary. One of the PCR based approaches for increasing ligation efficiency is to 
introduce new restriction sites into the PCR products through the use of mutagenic 
primers. In the modified restriction site PCR (MRPCR) approach (7, Table 1-2.1 and 
Fig. 1-2.1), polylinkers with a restriction site are ligated to the template and are then 
PCR amplified using different primers. One of the primers pairs with the polylinker, 
but bears an inactive restriction site. Another internal primer serves to introduce muta- 
tion into the construct at the expected site. Using this approach, new restriction sites 
are introduced into the ends of the final PCR products whereas mutations can be tar- 
geted anywhere in the construct. Likewise, new restriction sites can also be created at 
any internal position of the gene using internal mutagenic primers by the new restric- 
tion site PCR (NRPCR) approach (8, Table 1-2.2 and Fig. 1-2.2). These measures 
could improve post-PCR ligation, and therefore increase overall cloning efficiency. 
Because a restriction enzyme digestion is included, restriction sites on the primers are 
carefully chosen to avoid internal cleavage of the target sequence by the same restric- 
tion enzymes. 

In the case of circular templates, in vivo homologous recombination can be 
employed. Two different approaches have been developed for use with circular tem- 
plates. The so-called recombinant circular PCR (RCPCR)(9, Table 1-4.3 and Fig. 1- 
4.3) approach represents the combined use of OEPCR and IPCR. The second approach, 
recombinant PCR (RPCR) (9, Table 1-4.4 and Fig. 1-4.4), is a modified IPCR where 



172 Shen 

two overlapping primers are used, instead of end-to-end primers. The use of in vivo 
homologous recombination eliminates post-PCR processing and in vitro ligation, and 
therefore further improves the overall cloning efficiency. 

5. Selection for Desired Products 

Yet another advantage of the aforementioned MRPCR approach is that a restriction 
enzyme digestion adds a selection tool for the mutagenized products in the post-PCR 
process. By using carefully designed mutagenic primers, the only PCR products that 
can be digested and ligated into a vector are those amplified from primers bearing an 
activated restriction site. This added measure of selection helps eliminate the 
nonmutant background. 

Marker coupled PCR (MCPCR) approach (10, Table 1-3.1 and Fig. 1-3.1) is an 
example in which selection strategy is used for circular templates. An antibiotic resis- 
tance marker gene is targeted for selective activation in MCPCR instead of using 
restriction sites. For this approach to work, the template plasmid must have a point and 
null mutation in one of its otherwise functional antibiotic resistance gene. One of the 
PCR primers must be designed so that when incorporated into the new PCR product it 
restores the function of that marker gene. Following MCPCR, DNA products are trans- 
formed into E. coli. Transformants carrying desired mutagenized plasmids are then 
selected on media with the appropriate antibiotics. 

6. Random and Extensive Mutagenesis 

All of the approaches discussed so far are designed to create either single or a few 
site-directed mutations. Sometimes, it is desirable to obtain random and extensive 
mutations (REM) in the gene of interest or to generate a library of such molecules. 
There are two basic approaches to achieve these goals, and both are based on selected 
uses of PCR reaction components. The first approach (11) to generate REM makes 
use of error-prone DNA polymerases (Table 1-5.1 and Fig. 1-5.1). Certain thermo- 
stable DNA polymerases, like Taq DNA polymerase, have an intrinsic error rate due 
to the lack of a 3'-5' exonuclase activity. Each pass of the polymerase during PCR 
allows the possibility of mutations; therefore, the cumulative error rate can be sub- 
stantial. This tendency is further enhanced by other factors such as buffer composition 
(e.g., high-magnesium concentration, high pH, or addition of 0.5 mM MnCl 2 ) and 
other experimental conditions (e.g., a large amount of polymerase, a great number of 
cycles, a low-annealing temperature, a biased pool of the four dNTPs). 

The second approach (Table 1-5.2 and Fig. 1-5.2) to generate REM in the genes 
of interest is based on the base pairing property of degenerate base analog (12). For 
example, base analog dl can form base pairing with nucleotides A, C, G, and T 
under normal reaction conditions. In the presence of dl and a biased ratio of dNTPs, 
DNA polymerase tends to randomly incorporate a substantial amount of dl in the 
newly synthesized DNA strand. This dl-containing DNA can serve as a template in 
subsequent PCR amplifications and allows random base insertion at the dl-inserted 
sites. As a result, the final PCR products will have base substitutions at multiple and 
random sites. 



Mutagenesis and Recombination Overview 173 

Mutations at multiple but predetermined sites of a protein are sometimes desired. It 
is not difficult to see that the aforementioned approaches, when used singly, are not 
suitable for every need. Alternatively, stepwise elongation of sequence PCR (SESPCR) 
(13, Table 1-5.3 and Fig. 1-5.3) can be employed. In SESPCR, multiple mutagenic 
primers are utilized in a serial PCR to introduce mutations into the synthesizing gene. 
SESPCR is therefore practical only for relatively small genes. 

7. Creation of Novel Genes 

Our discussion has so far been focused on different PCR approaches for introduc- 
ing mutations (single or multiple and site-specific or random) into cloned genes. In 
fact, some of these basic approaches have also been modified to create novel genes or 
chimeric constructs from pre-existing mutations or genes through a process known as 
in vitro recombination. One of the approaches is Gene-Splicing Overlap Extension 
(Gene SOEing) (14, Table 1-4.1 and Fig. 1-4.1), which is an OEPCR-based approach. 
In Gene SOEing, two pairs of primers are used. One primer from each pair is actually 
a bipartite or modular primer with an oligonucleotide linker attached at the 5' end of 
the primer. The linkers from the two primer pairs are designed to overlap. When these 
two modular primers are from the same gene, Gene SOEing becomes OEPCR. Other- 
wise, if the two modular primers are based on DNA sequences from different genes, 
Gene SOEing produces a recombinant molecule. Such modular primer design allows 
virtually any two DNA sequences to be recombined. Consequently, Gene SOEing PCR 
is highly useful for in vitro gene fusion and protein engineering when appropriate 
restriction sites are not available at the expected sites of recombination. 

The second approach to in vitro DNA recombination is DNA shuffling or "sexual 
PCR" (15, Table 1-4.2 and Fig. 1-4.2). It is a modified megaprimer PCR. Basically, 
alleles of a target gene are mixed and fragmented by DNase I to different sizes (averag- 
ing 50-100 bp). These fragments serve as both primers and templates for PCR ampli- 
fications. Another slightly different modification is termed staggered extension primer 
(StEP) PCR (16, Table 1-4.2 and Fig. 1-4.2), which uses short random oligonuclotides 
to prime the synthesis of the new strand along the fragmented template. 

It is interesting to note that recombinant molecules generated by these two PCR 
approaches of in vitro recombination are dramatically different. In Gene SOEing, PCR 
products are not recombinants per se. When combined, melted, reannealed, PCR prod- 
ucts from different genes can form a heteroduplex intermediate as mediated by the 
overlapping linkers. Subsequent extension of the heteroduplex leads to the formation 
of recombinant molecules. Because linker primers define the recombination site, Gene 
SOEing-mediated DNA recombination is site-specific. A major application for this 
approach is in the study of gene fusion and protein domain swapping. In DNA shuf- 
fling and StEP, however, PCR products are already recombinant molecules. Recombi- 
nation happens during each PCR cycle when templates and primers switch following 
each denaturalization and reannealing process. As a result, the site of recombination is 
randomized and the recombination frequency is a factor of template length and exten- 
sion duration of each PCR cycle. PCR products generated in this manner represent a 
heterogenic mix of highly chimeric constructs. The resulting library of recombinant 
molecules is ideal for the study of in vitro evolution of enzymes. 



174 Shen 

We have briefly discussed some basic PCR approaches to DNA mutagenesis and 
recombination. Without going into much details of each approach, attempts have been 
made to present different approaches in a historical and logical prospective. In the 
postgenomic era, it is anticipated that PCR-mediated DNA mutagenesis and recombi- 
nation will become a common tool of molecular biologists, and hence, more innova- 
tive PCR approaches will certainly emerge over time. It is hoped that this review offers 
some guidelines for the reader to choose a PCR approach and to create additional 
innovations. 

References 

1. Ling, M. M. and Robinson, B. H. (1997) Approaches to DNA mutagenesis: An overview. 
Analyt. Biochem. 254, 157-178 

2. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G. and Erlich, H. (1986) Specific 
enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring 
Harbor Symp. Quant. Biol. 51, 263-273 

3. Kammann, M., Laufs, J., Schel, J. and Gronenborn, B. (1989) Rapid insertional 
mutageneisis of DNA by PCR. Nucl. Acids Res. 17, 5404. 

4. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R. (1989) Site-directed 
mutagenesis by overlap extension using the PCR. Gene 11, 51. 

5. Sarkar, G. and Sommer, S. S. (1990) The 'megaprimer' method of site-directed mutagen- 
esis. BioTechniques 8, 404. 

6. Ochman, H., Gerber, A. S. and Hartl, D. L. (1988) Genetic applications of inverse PCR. 
Genetics 120,621. 

7. Ito, W., Ishinuro, H. and Kurosawa, Y. (1991) A general method for introducing a series 
of mutations into cloned DNA using the polymerase chain reaction. Gene 102, 67 

8. Dulau, L., Cheyrou, A. and Aigle, M. (1989) Directed mutagenesis using PCR. Nucl. Acids 
Res. 17, 2873. 

9. Jones, D. H. and Winistorfer, S. C. (1991) Site-specific mutagenesis and DNA recombina- 
tion by using PCR to generate recombinant circles in vitro or by recombination of linear 
PCR products in vivo. Methods: Compan. Meth. Enzymol. 2, 2. 

10. Shen, T. J., Zhu, L. Q. and Sun, X. (1991) A marker-coupled method for site-directed 
mutagenesis. Gene 103, 73-77. 

11. Cadwell, C. and Joyce, G. F. (1992) Randomization of genes by PCR mutagenesis. PCR 
Meth. Appl. 2, 28-33. 

12. Spee, J. H.,de Vos W. M. and Kuipers, P. (1993) Efficient random mutagenesis method with 
adjustable mutation frequency by use of PCR and dITP. Nucl. Acids Res. 21, 777-778. 

13. Majumder, K. (1992) Ligation-free gene synthesis by PCR: synthesis and mutagenesis! 
at multiple loci of a chimeric gene encoding OmpA signal peptide and hirudin. Gene 
110, 89-94. 

14. Horton, R. M. (1997) In vitro recombination and mutagenesis of DNA: SOEing together 
tailor-made genes, in Methods in Molecular Biology, vol. 67, PCR Cloning Protocols: From 
Molecular Cloning to Genetic Engineering (White, B. A., ed.), Humana, Totowa, NJ, 
pp. 141-150. 

15. Stemmer, W. P. C. (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 
370, 389-391 

16. Zhao, H„ Giver, L., Shao, Z., Affholter, J. A. and Arnold, F. H. (1998) Molecular evolutions 
by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258-261. 



19 

In-Frame Cloning of Synthetic Genes Using PCR Inserts 
James C. Pierce 

1. Introduction 

Because many genes of biological interest are larger than the maximum size that 
current synthetic oligonucleotide synthesizers can produce (approx 110 bases), there 
is a need for methods that allow rapid production and expression of genes constructed 
from multiple synthetic DNA fragments. A number of synthetic genes have been gen- 
erated using the recursive polymerase chain reaction (PCR) method, but problems 
concerning primer design and incorrect final gene sequence, especially with large syn- 
thetic genes, are a concern (1-3). The cloning method described here follows a series 
of steps in which multiple PCR products or synthetic duplex oligonucleotides are posi- 
tionally cloned into a plasmid vector (4). A synthetic gene of practically any sequence 
or length can be built using the in-frame cloning method. Genes are assembled such 
that open reading frames are maintained by linking DNA fragments through the use of 
six basepair blunt-end restriction sites. Each cloning step uses an anchored sticky-end 
restriction site and a variable blunt-end restriction site that result in specific insert 
orientation and high cloning efficiencies. The overall strategy of in-frame cloning 
allows the researcher total control over nucleotide sequence, codon usage, promoter 
and other regulatory elements, and the placement of unique restriction sites throughout 
the recombinant construct. One advantage of the in-frame cloning method described 
here is that it allows for flexible yet precise construction of synthetic genes using 
standard recombinant techniques. Another advantage is that it employs inexpensive, 
readily available materials. 

The in-frame cloning method is based on the observation that standard plasmid 
cloning vectors such as pUC or pGEM contain very few six basepair blunt-end restric- 
tion recognition sites (5). The amino acid sequence of a protein, whose gene is to be 
cloned, is scanned for those amino acids encoded by the blunt-end restriction sites 
listed in Table 1. Each of these "signpost" amino acids can then be used to fragment a 
protein sequence into sections that are easily encoded by synthetic oligonucleotides. 
When two contiguous DNA fragments are joined in the plasmid vector by blunt-end 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

175 



176 Pierce 

Table 1 

Blunt-End Restriction Sites 

that Can be Used for In-Frame Cloning 



Enzyme 


Recognition Site 


Amino Acids 


Afel 


AGC-GCT 


Ser-Ala 


BsaAl 


CAC-GTG 


His-Val 


BsaAl 


TAC-GTA 


Tyr-Val 


Bstzni 


GTA-TAC 


Val-Tyr 


Btrl 


CAC-GTC 


His-Val 


EcoRY 


GAT-ATC 


Asp-Ile 


Fspa 


TGC-GCA 


Cys-Ala 


Hindi 


GTC-GAC 


Val-Asp 


Hindi 


GTT-AAC 


Val-Asn 


Hpal 


GTT-AAC 


Val-Asn 


Msd 


TGG-CCA 


Trp-Pro 


Nad 


GCC-GGC 


Ala-Gly 


Nrul 


TCG-CGA 


Ser-Arg 


Pmll 


CAC-GTC 


His-Val 


Pvull 


CAG-CTG 


Gin-Leu 


Seal" 


AGT-ACT 


Ser-Thr 


Sfol" 


GGC-GCC 


Gly-Ala 


Smal 


CCC-GGG 


Pro-Gly 


SnaBl 


TAC-GTA 


Tyr-Val 


SspV 


AAT-ATT 


Asn-Ile 


Stul 


AGG-CCT 


Arg-Pro 



"These restriction enzyme recognition sites are 
present at least once outside of the multiple cloning 
regions of pUC and pGEM vectors. Underlined codons 
can be problematic in E. coli expression systems. 

ligation the open reading frame of the synthetic gene is maintained. As described later, 
a combination of relatively simple cloning techniques and electroporation give high 
overall cloning efficiencies. The PCR is used for both synthesis of duplex DNA from 
oligonucleotides and for the rapid screening of intermediate and final synthetic plas- 
mid constructs by direct amplification of plasmid DNA from transformed host bacte- 
ria (Escherichia coli) colonies. By monitoring the final plasmid-synthetic gene 
construct for the presence or absence of diagnostic restriction sites, one can have good 
confidence that the correct synthetic gene has indeed been cloned before confirmation 
by DNA sequencing. 

The in-frame cloning method was initially developed to make synthetic genes from 
complementary synthetic oligonucleotides that were annealed to generate a duplex 
DNA molecule and then directly cloned into the plasmid vector (4). One advantage of 
using synthetic oligonucleotides as DNA fragments for direct cloning is the ability to 
use them without further purification. Also, because oligonucleotides do not contain a 
terminal phosphate group, no problems are encountered with multiple tandem copies 



In-Frame Cloning of Synthetic Genes 1 77 

of the DNA insert following ligation reactions in which the insert is in significant 
molar excess relative to the plasmid scaffold. The protocol described here is based on 
the work of a number of researchers in which duplex insert DNA is first made by the 
PCR using overlapping oligonucleotide primers (1,6,7,8). This modification allows 
for larger blocks of insert DNA to be made per cloning event and will decrease the 
overall cost of oligonucleotide primers. Two problems associated with PCR-derived 
templates are poor DNA sequence fidelity and difficulty in obtaining flush blunt-ends. 
With the availability of thermostable DNA polymerase with high replication fidelity 
and 3'-5' exonuclease activity that removes terminal sequences, many of the problems 
associated with poor-quality PCR derived templates can be overcome. 

2. Materials 

1. Plasmid vector DNA (e.g., pUC orpGEM). 

2. Synthetic oligonucleotides designed using the format in Fig. 1. Resuspend deprotected 
oligonucleotides in sterile water or Tris-ethylenediaminetetraacetic acid (EDTA) buffer, 
pH 8.0 at a final concentration of about 2 mg/mL. There is no need to purify oligonucle- 
otides as long as the synthesis was relatively efficient. The quality of the oligonucleotides 
can be checked by simple agarose or acrylamide gel electrophoresis and staining with 
ethidium bromide. 

3. E. coli host strain for plasmid transformation (e.g., strain DH10B, Life Technologies, 
Rockville, MD). 

4. Restriction enzymes and buffers. 

5. Reagents for agarose gel electrophoresis including low-melting point agarose for in-gel 
cloning (e.g., GTG SeaPlaque agarose, FMC BioProducts, Rockland, ME). 

6. DNA ligase and buffer. 

7. Microdialysis membrane such as Millipore VSP 0.025-mm filters (Bedford, MA). 

8. A high efficiency method to transform E. coli cells with plasmid DNA (e.g., Bio-Rad 
Gene Pulser electroporator, Hercules, CA). 

9. Media, Petri plates, and ampicillin for growth and selection of transformed bacteria. 

10. PCR primers that border the cloning site of the plasmid vector (e.g., Sp6 and T7 primers 
are used for pGEM vectors). 

11. Reagents and thermocycler for PCR. 

12. Reagents needed for alkaline lysis, miniplasmid purification procedure (9). 

13. DNA sequencing reagents and equipment for final confirmation of synthetic gene 
sequence. 

14. TE buffer: 10 mM Tris-HCl, pH 8.0, 1 mM ethylediaminetetraacetic acid (EDTA). 

15. TBE buffer: 90 mM Tris, 90 mM borate, pH 8.3, 2 mM EDTA. 

16. TE-saturated phenol. 

17. Chloroform:isoamyl alcohol (24:1). 

18. 95% ethanol. 

19. 70% ethanol. 

3. Methods 

3.1. Organizational Strategy for Oligonucleotide Design 

1. The in-frame cloning method assumes that the amino acid sequence of the protein is 
known for the synthetic gene under construction. Plasmid vectors such as pUC and pGEM 



178 



Pierce 



NH2. 



PROTEIN 



_COOH 



12 3 4 5 6 



34 35 36 37 38 39 40 . 



67 68 69 70 71 72 73 



i 



e.g.) amino acid #37 = alanine 





A 


PCR Strategy 






F«¥;¥; 


1- 


c 






I.:.}:.:-:-:-:-:-:.:.:::-:.:.:.:-:-:-:-:-:.::.::-:-:-::-:::-: 


:■:■:■:■:-, 






1 

D 


mmm 





L 



Template for PCR Fragment 



3' CASSETTE 



5' 


CODING SEQUENCE 


X 


Y 


Z 



EcoRI restriction site or 
blunt end 



Blunt end restriction site I 

Diagnostic restricion site 

Hindlll restriction site. 



EcoRI 
1 1 


PCR Fragment A/B 


A fl" Hindlll 
Noel 1 1 


1 l| 1 1 1 
GCC GGC CT] AAGCIT TGA 








1 

Start codon 




1 
alanine #37 



Fig. 1. Design strategy for the in-frame cloning method using PCR-generated DNA inserts. 
The design strategy is broken into four parts. Part 1 illustrates the analysis portion of the in- 
frame cloning procedure. The primary amino acid sequence is scanned for amino acids that are 
present in Table 1. The protein sequence is divided into segments that are encoded by overlap- 
ping synthetic oligonucleotides (Part 2). In this example, amino acid #37 is an alanine, which 
defines the end point between segment 1 (encoded by oligos A/B) and segment 2 (encoded by 
oligo C/D). The PCR is used to extend the 3' end of each oligonucleotide. Part 3 demonstrates 
the template from which to design the synthetic oligonucleotides. Each PCR-generated frag- 
ment contains three main components: the 5'end, the coding sequence and the 3' cassette. The 3' 
cassette is further divided into three sections. Section X represents the blunt-end restriction site 
picked from Table 1, section Y represents the diagnostic restriction site for segment identifica- 
tion, and section Z represents the 3' cloning site, usually a Hindlll sticky-end. Part 4 shows the 
partial sequence of PCR fragment A/B. 



In-Frame Cloning of Synthetic Genes 1 79 

are used as the scaffold to build the synthetic gene. These plasmids have relatively few 
six base-pair blunt-end restriction recognition sites (listed in Table 1). Seventeen of the 
20 naturally occurring amino acids are available using the restriction sites listed in 
Table 1. Note that some of the codons illustrated in Table 1 have been identified as being 
problematic when used in E. co/i'-based recombinant expression experiments (10). The 
in-frame cloning method uses this knowledge to design a cloning strategy that allows the 
forced orientation cloning of insert DNA such that the open reading frame is maintained 
by the assembly of DNA fragments at one endpoint by blunt-end ligation and at the other 
endpoint by sticky-end ligation (for overview, see Figs. 1 and 2). 

2. A number of symmetrical six base-pair blunt-end restriction sites, listed in Table 1, can 
be used for in-frame cloning. The protein is broken into a number of fragments that con- 
tain one of the amino acids listed in Table 1. These selected amino acids define the last 
amino acid of a protein segment in each of the cloning steps. It is possible to build a gene 
starting from either the amino terminus or the carboxyl terminus. This gives more flex- 
ibility in the choice of amino acids that are encoded by the blunt-end restriction sites. 
Once the protein sequence has been segmented into specific insert blocks (see Fig. 1, part 
1 and 2), a PCR strategy is utilized which will result in DNA fragments that contain all 
the elements needed for in-frame cloning. 

3. Fig. 1 illustrates the design strategy for generating PCR fragments that are used in the 
in-frame cloning method. The protein sequence is segmented into blocks that are easily 
covered by moderate sized oligonucleotides of about 50-90 bases. Part 1 of Fig. 1 shows 
a hypothetical 73 amino acid protein that will be broken into two parts between amino 
acids #37 and #38. Note that amino acid #37 is alanine, which is encoded by one or more 
of the blunt-end restriction enzymes listed in Table 1. The oligonucleotides are designed 
such that a 20 base-pair region of complementary DNA sequence overlap will result when 
the two cognate oligonucleotides are annealed together (e.g., oligos A and B in part 2 of 
Fig. 1). As an example, if two 80 base oligos were annealed together with a 20 base 
overlap, a protein coding sequence of up to 46 amino acids could be generated. Once 
annealed, duplex DNA is made by extension of the 3' end of each oligo using PCR for a 
limited number of cycles (see Note 1). 

4. The PCR fragment must contain a number of elements as illustrated in section 3 of Fig. 1. 
The 5' end contains either an EcoRl restriction site or a blunt-end. The EcoSl site is used 
during the first round of cloning. Subsequent rounds of cloning use a 5' blunt-end, which, 
upon ligation to the 3' blunt-end generated during the cloning process, will result in the 
correct translational reading frame. The 5' blunt end can be made by either of two (different) 
methods. If a DNA polymerase is used that generates flush blunt ends during the amplifica- 
tion process, then oligonucleotides can be designed such that the 5' end of the fragment 
(first three nucleotides) will contain the next codon in the amino acid sequence. If there is 
uncertainty concerning the sequence (e.g., terminal synthetase activity) at the 5' (left) end, 
then it would be necessary to generate an oligonucleotide that contains an internal blunt-end 
recognition site which upon restriction digestion of the PCR fragment with the appropriate 
enzyme will leave a suitable blunt-end. This second scenario is somewhat restrictive in that 
the amino acid must be encoded by a symmetrical six-base blunt-end restriction enzyme 
(not necessarily from Table 1) and makes the design of oligonucleotides more difficult. The 
internal part of the oligonucleotide contains the nucleotide sequence information that 
encodes the amino acids of the protein. The 3' end contains a number of restriction sites and 
is termed the 3' cassette. As shown in Fig. 1, part 3, the 3' cassette contains three sites 
labeled X, Y, and Z. Site X contains the blunt-end restriction site that will align the amino 



180 



Pierce 




EcoRI Hindlll 



- digest plasmid with EcoRI and Hindlll 

- purity plasmid on LMP gel 

- ligate plasmid and duplex PCR fragment in gel 

- transform host cell and select for plasmid 

- screen colonies by direct PCR for fragment insert 

- prepare plasmid DNA for the next round of In-frame cloning 




- digest plasmid (insert #1 ) DNA with Nael and Hindlll 

- follow cloning protocol as above 




- digest plasmid (insert #1 , #2) with Nael and Hindlll 
■ follow cloning protocol as above 



Plasmid contains 
the complete 
in-frame coding 
sequence of 
synthetic gene. 




- Repeat cloning procedure if more 
PCR fragments are needed 
to generate synthetic gene. 



Fig. 2. Implementation of the in-frame cloning method for a three-insert synthetic gene. The 
execution of the cloning strategy is broken into four parts, 5-8. Part 5 shows the cloning of 
PCR insert #1 into a standard plasmid vector (e.g., pUC). This duplex PCR-generated segment 
is cloned as an £roRI///;'fldIII fragment. The steps used in the in-frame cloning protocol are 
listed in sequential order next to the plasmid diagram. Part 6 illustrates the cloning of PCR 
insert #2 (blunt-end/////?dIII fragment). Part 7 illustrates the cloning of PCR insert #3 (blunt- 
end/Hindlll fragment). Part 8 shows the completed plasmid construct containing the synthetic 
gene. The bold arrow represents the ability to continue to clone as many DNA inserts as are 
needed to complete an open reading frame for any synthetic gene. 



In-Frame Cloning of Synthetic Genes 181 

acids of the open reading frame upon blunt-end ligation. Site Y contains a diagnostic 
restriction site used to monitor placement of the insert when screening plasmid clones. 
Site Z contains the Hindlll restriction site that is used to anchor the 3' DNA fragment to 
the plasmid during each ligation step of the cloning process. 

5. Figure 1, part 4 illustrates the partial sequence of PCR fragment A/B that has been gen- 
erated using the template from Fig. 1, part 3. This fragment contains a 5' EcoRl cloning 
site, a translational start codon, a Nael blunt-end site, an Aflll diagnostic site, and a 3' 
Hindlll cloning site. After restriction digestion with EcoRl and Hindlll, this fragment 
can be cloned into the corresponding sites of the plasmid vector. Note that after cloning 
this fragment into the plasmid vector, cleavage with the restriction enzyme Nael will 
generate a blunt-end which encodes the GCC codon of alanine (amino acid #37) at the 3' 
terminus. As shown below (see Fig. 2), subsequent rounds of in-frame cloning will insert 
the 5' blunt-end of the incoming fragment (C/D) next to the 3' AWI-generated blunt-end 
of the first insert (A/B). Assuming the first three basepairs of fragment C/D encode amino 
acid #38, the translational reading frame of the synthetic gene will be maintained. 

6. The design of primers for PCR fragment synthesis must be carefully monitored before 
oligonucleotide synthesis. Three points are worth noting: First, make sure that the inad- 
vertent engineering of necessary unique restriction sites in other parts of the fragment 
does not occur. Screen the oligonucleotide sequence for all six-base restriction recogni- 
tion sites before synthesis. It is important that the EcoRl, Hindlll, blunt-end, and diag- 
nostic restriction sites all remain unique to the engineered cloning vector. Second, make 
sure that the restriction sites needed for moving the synthetic gene from one vector to 
another have been correctly engineered. In most cases, the synthetic gene will be engi- 
neered in a standard plasmid vector (e.g., pGEM or pUC) and then subcloned into an 
expression vector (e.g., pMAL, baculovirus, and so on). Finally, remember to include all 
regulatory elements such as ribosome binding sites, transcription terminator sequences, 
and so on, if these elements are not present in the expression vector which the synthetic 
gene is to be cloned into. 

3.2. Construction of a Synthetic Gene Using In-Frame Cloning 

1 . Figure 2 illustrates the in-frame cloning process once an insert fragment has been gener- 
ated. The protocol described in this review uses PCR generated fragments but synthetic 
duplex oligonucleotides or other cloned plasmid-derived fragments could also be used. 
The first round of in-frame cloning uses a PCR insert, which has EcoRl and Hindlll 
sticky-ends at the 5' (left) and 3' (right) ends, respectively. All subsequent rounds use a 5' 
blunt-end and 3' Hindlll sticky-end duplex DNA fragments as inserts. By a reiterative 
process of cutting the plasmid vector with iicoRI/blunt-end and Hindlll restriction en- 
zymes followed by agarose gel purification and ligation, a synthetic gene of almost any 
length can be constructed. The only limiting factor is the availability of appropriately 
spaced single amino acids in the protein sequence that are encoded in the blunt-end re- 
striction sites listed in Table 1. Screening for plasmids with successful ligation of insert 
DNA is done by direct PCR analysis of antibiotic resistant bacterial colonies. This method 
greatly simplifies the isolation of positive clones and allows for the rapid construction of 
moderate to large-sized synthetic genes. 

2. Assemble the following for a standard 40 (xL PCR: 0.5-1.5 (ig primer A (approx 30 pmol 
of a 60-80 base oligo), 0.5-1.5 jig primer B (approx 30 pmol of a 60-80 base oligo), 4 (xL 
10X buffer, 4.0 \xL 25 mM MgCl 2 , 4.0 u.L of 2 mM dNTPs, 1 U Taq polymerase, and 
water to 40 uL total volume. 



182 Pierce 

3. Perform PCR using the following conditions (see Note 1): 94°C for 1 min (denaturation); 
55°C for 2 min (annealing); 72°C for 2 min (extension) for five cycles; and 72°C for 10 min 
(final extension). 

4. If a mineral oil overlay was used add an equal volume of chloroform, mix and transfer the 
aqueous layer to a sterile microcentrifuge tube. Incubate the tube with the lid open at 
37°C for 15 min or until there is no trace odor of chloroform. 

5. While the sample is drying, fill a 100-mm sterile Petri plate with 35 mL of IX TE buffer 
(see Note 2). Float a Millipore VSM 0.025-u.m filter (shiny side up) on the TE buffer. 

6. Place the PCR amplified DNA sample (up to 100 uL) on the filter and allow 30-60 min 
for dialysis. Remove the sample and place it in a sterile microcentrifuge tube. 

7. Digest an aliquot of the PCR amplified DNA sample by adding the following reagents to 
a microcentrifuge tube and incubating at 37°C for 1 h: 16 [xL DNA sample, 2 [xL 10X 
buffer, 1 (xL Hindlll and 1 [xL EcoKl. 

8. After the digestion is complete, purify the PCR DNA fragment away from the released 
cut ends (see Note 3). The protocol listed here is based on the Qiagen QIAquick-spin 
PCR purification columns. For more details, see manufacturer's product description. 

9. Following restriction digestion add 0.5 mL of binding buffer to DNA sample. Apply 
sample to QIAquick-spin column and centrifuge column in a 2-mL centrifuge tube for 60 s 
at maximum speed. Remove flowthrough and wash column with 0.75 mL of wash buffer 
as above. Remove flowthrough and centrifuge again for 60 s to remove residual wash 
buffer. Elute DNA by adding 50 [xL of TE buffer to the spin column, placing the spin- 
column in a sterile 1.5-mL microcentrifuge tube and centrifuging for 60 s at maximum 
speed. The DNA fragment is now ready to be cloned into the plasmid cloning vector. 

10. Plasmid vector DNA is prepared by cutting with restriction enzymes in the following 
manner: 1-2 [xg plasmid DNA (e.g., pGEM), 2 uL 10X buffer, 1 \xLEcoRl, 1 [ih Hindlll, 
and water to 20 u,L total volume. Incubate restriction digest at 37°C for 1 h. 

1 1 . The cut plasmid DNA is purified away from the small DNA fragment released during the 
double digest using low melting point (LMP) agarose gel electrophoresis (see Note 4). 
The purified DNA fragment is then ligated to the PCR insert fragment that was purified 
in step 9. Add to a microcentrifuge tube the following components: 5 (xL purified PCR 
fragment (approx 0.5 (xg), 10 jxL plasmid DNA fragment in agarose plug (50-100 ng), 
4 (xL 5X ligation buffer, and 1 (xL DNA ligase. The ligation reaction is incubated over- 
night at room temperature (see Note 5). 

12. Transform appropriate E. coli host cell (e.g., strain DH10B from Life Technologies) by 
electroporation or other high efficiency transformation protocol. Before electroporation, 
remove the salt from the ligation reaction by dialysis against 0. IX TE buffer for one hour 
at room temperature (see Note 2). 

13. Electroporation using a Bio-Rad E. coli Gene Pulser (Hercules, CA) is performed as 
follows. The agarose plug containing the ligation reaction is melted at 70°C for 10 min 
and 5 [xL of the molten reaction is added to 80 u.L of electrocompetent host cells (E. coli 
strain DH10B). The DNA/host cell mixture is then placed in a 0.1-cm cuvet and 
electroporated at a setting of 1.80 kV. Add 1 mL of liquid media (e.g., L broth) and 
incubate with shaking at 37°C for 1 h. One-tenth of the transformed cells are then spread on 
a LB agar selector plate containing 50 mg/mL ampicillin. The plates are then incubated 
overnight at 37°C. 

14. Screening for positive PCR fragment inserts is performed by direct PCR analysis of colo- 
nies from the ampicillin selector plates. An agar plug (see Note 6) of an individual 
ampicillin resistant colony is resuspended in 50 u,L of sterile water and mixed well. To 



In-Frame Cloning of Synthetic Genes 183 

each 40 (xL PCR sample tube add the following reagents: 5 jxL agar plug colony mixture 
(DNA template), 100 ng (ca. 15 pmol) each forward and reverse pUC/M13 primers (or T7 
and Sp6 for pGEM vectors), 4 uE 10X buffer, 4 [xL 25 mM MgCl 2 , 4.0 uE 2.0 mM dNTPs, 

1 U Taq polymerase, and water to 40 [xL final volume. 

15. Perform PCR using the following conditions: 94°C for 1 min (denaturation), 55°C for 

2 min (annealing), and 72 C C for 2 min (extension), 30 cycles. 

16. Analysis of PCR samples is performed by agarose gel electrophoresis using 0.5 X TBE 
buffer. To observe DNA fragment sizes in the range of 100 to 300 basepairs, a 3% agar- 
ose gel fractionates well. Bacteria that contain clones that are positive for the correctly 
ligated DNA insert will contain a PCR DNA fragment that is larger than those PCR DNA 
fragments amplified from bacteria that contain only vector DNA (see Note 8). 

17. Once positive colonies have been identified, plasmid DNA is prepared by the alkaline 
lysis or other DNA mini-prep procedures (9,11). 

18. To confirm that the PCR fragment has been cloned into the plasmid vector a restriction 
digest is performed. The choice of restriction enzyme depends upon which diagnostic site 
was engineered into the DNA insert (see Fig. 1, part 3). In addition to the insert diagnos- 
tic restriction enzyme, use restriction enzymes that will reveal correct insert ligation and 
the removal of the plasmid vector multiple cloning region (see Note 9). To a 
microcentrifuge tube add the following reagents: 5 (xL plasmid DNA (approx 0.2 fxg), 
2 uE 10X restriction buffer, 1 [xL restriction enzyme, (e.g., AfllT) and water to 20 (xL total 
volume. Incubate the restriction digest(s) at the appropriate temperature for 1 h. 

19. Fractionate the plasmid DNA from the restriction digests by agarose gel electrophoresis 
using a 0.8 % gel and 0.5X TBE buffer. A plasmid clone that is positive for the correct 
DNA insert will have a restriction pattern that indicates the presence of the diagnostic 
restriction site (e.g., Aflll). Once a positive clone is identified by PCR and then confirmed 
by restriction mapping, the next round of in-frame cloning is initiated. The next round of 
in-frame cloning (see Note 10) proceeds with a ligation reaction using the blunt-end and 
Hindlll cut plasmid (insert 1) that was engineered in the first round of in-frame cloning 
(see Fig. 2, part 5), and the PCR fragment generated from oligonucleotides C and D (see 
Fig. 1, part 2). 

20. Using the plasmid DNA prepared in the mini-prep procedure (step 17) from one of the 
positive clones, perform a restriction enzyme double digest. One restriction enzyme will 
be Hindlll. The second restriction enzyme will be a six basepair, blunt-end recognition 
site enzyme listed in Table 1. If the buffer and/or temperature conditions are incompat- 
ible for both enzymes together in one reaction, then perform the digest sequentially. To a 
microcentrifuge tube add the following: 1 (xg plasmid DNA (containing insert 1), 2 (xL 
10X buffer, 1 fxL Hindlll, 1 (xL blunt-end restriction enzyme from Table 1, and water to 
20 [xL total volume. Incubate restriction enzyme at the appropriate temperature for 1 h. 

21. Purify the cut plasmid (insert 1) away from the small DNA fragment released during the 
double restriction digest using low-melting agarose gel electrophoresis exactly as 
described in step 11 and Note 4. 

22. Prepare the second DNA insert (insert 2) by following the protocol outlined in steps 1-9. 
It is generally more efficient to prepare all of the DNA inserts (e.g., insert 1, insert 2, and 
so on) at the same time. After PCR extension to generate the double-stranded DNA frag- 
ment, the fragment must be digested with Hindlll. As mentioned in Subheading 3.1., 
part 4, if blunt-ends are difficult to achieve during the PCR, then a second restriction 
digest must be performed to generate the correct fragment ends needed for ligation. Per- 
form the Hindlll digest as follows: 17 [xL DNA sample, 2 u.L 10X buffer, and 1 [xL 



184 Pierce 

Hindlll. Incubate the reaction at 37°C for 1 h. Purify the DNA fragment away from small 
released fragment(s) by column chromatography as described in steps 8 and 9. 

23. A ligation reaction is prepared exactly as described in step 11. After ligation and transfor- 
mation (steps 11-13), positive clones are identified as described in steps 14-19. In-frame 
cloning can be reiterated as many times as is necessary to engineer a synthetic gene of the 
desired length (see Fig. 2, part 8). 

24. When all of the necessary rounds of in-frame cloning have been completed, the insert nucle- 
otide sequence must be confirmed by DNA sequence analysis. Candidate plasmid constructs 
should first be confirmed by analysis of the correct insert size (as determined by PCR ampli- 
fication and comparison to vector-with-no-insert and intermediate plasmid constructs) and 
by restriction mapping to indicate that the correct diagnostic restriction sites are present 
and that certain vector restriction sites have been removed. Sequence analysis can be per- 
formed by double stranded PCR sequencing using Ml 3/pUC universal primers (11). Single- 
stranded DNA can be made and sequenced using vectors such as the pGEM-fl ori series. 

25. A rough timeline is presented below to serve as a guide for experiment design. It is 
assumed that all oligonucleotide design and synthesis has been completed. It also assumes 
that a preliminary PCR experiment has shown that the oligonucleotide PCR fragments 
are of the correct size and of decent quality. 

Day 1 : PCR amplification of oligonucleotide insert fragments 

Gel elelectrophoretic analysis of PCR samples 
Day 2: Cut PCR fragments with restriction enzyme(s) 

Cut plasmid vector with restriction enzymes 

Purify PCR fragments by spin chromatography 

Purify plasmid vector by LMP agarose gel electrophoresis 

Set up ligation reactions and incubate overnight 
Day 3: Transform E. coli host cells 

Grow colonies overnight on ampicillin selector plates 
Day 4: Screen selected colonies by direct PCR 

Gel electrophoresis of PCR/colony samples 

Overnight culture of tentatively positive colonies 
Day 5: Miniprep plasmid DNA isolation 

Restriction mapping using diagnostic site analysis 

Gel electrophoresis of plasmid restriction digests 

Prepare vector for the next round of in-frame cloning 

4. Notes 

1. The generation of the duplex DNA fragments in this protocol is really just a DNA poly- 
merase extension reaction. The PCR is used because it is simple and the reagents are 
generally available. A small number of amplification cycles (e.g., 5) should generate suf- 
ficient product. If priming artifact occurs try using fewer cycles. One cycle may be enough 
to generate sufficient duplex DNA for cloning. If PCR product artifacts are a problem, the 
use of 5' outside primers to amplify the correct size duplex fragment from the initial PCR 
amplification may be needed. The use of synthetic oligonucleotide duplex DNA for direct 
insert cloning is always an option. 

2. The author uses microdialysis routinely to prepare DNA substrates that must undergo a 
number of different and separate enzymatic treatments. Although many DN A/enzyme 
reactions work adequately under "universal" buffer conditions, complex protocols that 
require a number of linked reactions often show low overall efficiency. It has been my 



In-Frame Cloning of Synthetic Genes 185 

experience that if each DNA modification reaction is treated individually, the success of 
the entire experiment is greatly enhanced. Microdialysis is a technique that allows the 
efficient exchange of buffers for any DNA sample. Once mastered it is often more conve- 
nient and efficient than ethanol precipitation or spin chromatography. The following pro- 
tocol works well for DNA fragments 100 bases or larger. 

a. Fill a 100-mm sterile Petri plate with 35 mL of IX TE buffer. Float a Millipore VSM 
0.025-mm filter (shinny side up) on the TE buffer. Make sure no buffer wets the top 
side of the filter membrane. 

b. Place the DNA sample on the filter. Volumes from 10-100 uL can be used but care 
must be taken with larger volumes. Multiple samples can be placed on one filter as 
long as they do not contact one another. As described later, sample volumes often 
increase or decrease, so beware! 

c. Place the lid on the Petri plate and incubate for about one hour at room temperature. 
Make sure the Petri plate is in a safe place on the bench because if it is disturbed, the 
sample may be lost. Length of time will depend on how dramatic the change in buffer 
conditions will be. Simple desalting can usually be accomplished in less than 30 min. 

d. Remove the sample using a pipeting device and place it in a sterile tube. The volume 
of the DNA sample can change significantly because of osmotic imbalances or evapo- 
ration, so it is often useful to record the pre-and post-microdialysis volumes. It has 
been my experience that very little DNA is lost by non-specific binding to the mem- 
brane in this procedure. 

e. This protocol works well with agarose plugs when it is necessary to remove the elec- 
trophoresis buffer before enzymatic manipulation. The agarose plug is melted at 70°C 
and then placed directly on the filter that is floating on the TE buffer. The molten 
agarose will form a semi-solid plug on the filter but this does not interfere with buffer 
exchange. To remove the agarose plug containing the DNA sample, first lift the entire 
filter membrane off the buffer using forceps. Then scrape the agarose plug off the 
filter directly into a microcentrifuge tube using a sterile scalpel. The sample is then 
incubated at 70°C and the agarose-DNA sample can then be added to the next reac- 
tion mixture. 

3. Ligation reactions are not always logical! Often the products recovered from a ligation 
reaction are a mixture of intended constructs and a collection of obscure and unlikely 
side-products. The source of unintended DNA constructs is often insert-insert ligation 
events. Therefore, it behooves one to think critically about just what DNA substrates are 
being placed in the ligation reaction. When synthetic duplex oligonucleotides are used as 
DNA inserts, there is not much concern about insert ligating to itself since the 5' ends are 
not phosphorylated. With the PCR generated DNA fragment approach described in this 
protocol, self-ligation is a potential problem. Although the forced orientation cloning 
approach minimizes ligation artifacts, there is still the possibility of multiple inserts being 
cloned into the vector during a ligation reaction. 

The first round of in-frame cloning uses an EcoRl/Hindlll sticky-ended fragment. 
There is significant possibility for self-ligation under the conditions used here (see later). 
Subsequent rounds of in-frame cloning using blunt-end/H;«dIII substrates pose less of a 
problem because of the lower efficiency of blunt-end ligation. A tripartite £'coRI///»7dIII 
fragment will insert into the plasmid vector giving an incorrect construct. Because the 
in-frame cloning method screens for positive DNA inserts using direct PCR amplifica- 
tion of selected colonies followed by fragment size analysis using gel electrophoresis, 
this multiple insert problem should not interfere with the engineering experiment. 



186 Pierce 

4. The double-digested plasmid is purified on a 0.6% LMP agarose gel using 0.5X TBE 
buffer. The gel is stained with ethidium bromide and the linearized plasmid cut out and 
then placed in 1 mL of sterile water to remove excess TBE buffer. After 30 min, remove 
the water and heat the agarose gel plug at 70°C for 10 min. It is important that the compo- 
nents of next reaction have already been prepared before the agarose gel fragment is 
melted. After heating, the molten agarose is added directly to the ligation reaction before 
it solidifies. Using the pipet tip, gently mix the molten agarose with the ligation reaction 
components and allow the tube to sit at room temperature. Do not centrifuge the sample 
once the agarose has been added, as this will separate the plug from the other reaction 
components. Depending on the reaction buffer and the amount of agarose added, the reac- 
tion mixture may or may not solidify at room temperature. 

5. The molar ratio of insert to vector in the ligation reaction described in this protocol is 
approx 100:1. This is calculated by assuming the plasmid vector to be 3000 bp of which 
0.1 (xg is added to the ligation reaction. This would equal about 0.05 pmole of DNA (1 bp 
= 660 Daltons, 1 pmole of a basepair of DNA = 660 pg). If the average insert fragment is 
about 150 bp and 0.5 |xg were used in the ligation reaction then about 5 pmoles would be 
present in the sample. Thus, a 5-0.05 pmole DNA ratio is achieved. This ratio is some- 
what high for a standard sticky-end ligation reaction but is used to drive the ligation 
reaction in the agarose plug. If problems with multiple inserts occur, then decrease the 
concentration of fragment insert. It is difficult to modulate the concentration of vector 
DNA because an agarose plug strategy is being used and low cloning efficiencies are a 
problem if too little plasmid vector is used during transformation. In general, the condi- 
tions described here work well and the majority of plasmids contain the correct insert. 
Forced orientation and removal of restriction cleavage ends "push" the ligation reaction 
in the direction of correct insertion. 

6. Screening for positive vector inserts is best accomplished by direct PCR amplification of 
bacterial colonies taken from the ampicillin selector plates. A sterile 50-mL capillary 
pipet (or 1-mL pipet) is used to isolate or "plug" well-separated colonies from the agar 
plate. The agar plug is then placed in a sterile 1.5-mL microcentrifuge tube containing 
50 |iL of sterile water and mixed well using a vortex mixer. A 5-u.L sample of this bacte- 
rial colony/agar plug/water mixture is then added to a standard 40 (xL PCR tube as 
described in the protocol. The author has found it convenient to work-up 18 samples at a 
time and then perform gel electrophoretic analysis using a 20-well comb with two lanes 
used as molecular weight marker and control PCR (vector no-insert), respectively. The 
tubes containing the bacterial colony/agar plug are stored at 4°C until the PCR and elec- 
trophoresis results are obtained. Once positive colonies are tentatively identified, an 
overnight culture is made using 3 mL of Luria broth, 50 mg per mL of ampicillin, and 
25 uL of the bacterial colony/agar plug sample. This is grown overnight at 37°C with 
moderate shaking. The overnight culture can then be used to make an alkaline lysis 
miniprep DNA sample that is then used for restriction mapping and/or prepared for the 
next round of in-frame cloning. Once positive constructs are identified, the remaining 
overnight culture can be kept for long term storage by adding glycerol to 30% of total 
volume, mixing well and then storing at -80 C C. 

7. These primers flank the multiple cloning site of many pUC-based cloning vectors. If 
another vector is being used for in-frame cloning you may need a different set of primers. 
A control reaction should be performed from a cell-containing vector DNA that has not 
been manipulated (usually taken from the selector plate used to monitor transformation 
efficiency, e.g., uncut pGEM DNA). 



In-Frame Cloning of Synthetic Genes 187 

8. The difference in fragment size between the control PCR sample (colonies with vector-no 
insert) and experimental samples (colonies with vector plus insert) will allow you to deter- 
mine which host cells contained plasmid DNA with the PCR fragment insert cloned into 
the EcoRl/Hindlll site. Remember to subtract the length of the multiple cloning region 
(approx 50 bp) of uncut pGEM or pUC-based vectors when comparing fragment sizes. 

9. Analysis of the diagnostic restriction site (e.g., Aflll) as an identifier for the correct insert 
fragment, and loss of restriction sites (e.g., Pstl) from the multiple cloning region (MCR) 
of the vector, ensures that this PCR fragment was cloned and the vector MCR removed. 
You can also check for the regeneration of the £roRI and Hindlll sites. This restriction 
digest screen will allow you to have good confidence that the correct fragment has indeed 
been cloned. During this restriction analysis the plasmid DNA can be prepared for the 
next step of in-frame cloning by cutting with Hindlll and the blunt end recognition restric- 
tion enzyme. All of these samples can be run on a low-melting-point agarose gel and the 
////7dIII/blunt end cut plasmid purified as described above (also see Note 2). 

10. This process can be reiterated as many times as is necessary to clone a moderate to large- 
sized synthetic gene (200-1000 bp). If a very large gene is going to be made by the 
synthetic in-frame cloning method it may be more practical to use two or more plasmids 
as the templates for constructing different portions of the gene. Just duplicate each of the 
cloning and processing steps using different DNA inserts. The assembled gene fragments 
are then isolated by blunt-end/blunt-end or blunt-end///z'/?aTII restriction digestion and 
then linked to the primary plasmid template by the in-frame cloning process. 

References 

1. Prodromou, C. and Pearl, L. H. (1992) Recursive PCR: a novel technique for total gene 
synthesis. Protein Eng. 5, 827-829. 

2. Jaffe, E. K., Volin, M., Bronson-Mullins, C, Dunbrack, R. L., Kervinen, J., Martins, J., et al. 
(2000) An artificial gene for human porphobilinogen synthase allows comparison of an allelic 
variation implicated in susceptibility to lead poisoning. /. Biol. Chem. 275, 2619-2626. 

3. Johnson, T. M., Quick, M. W., Sakai, T. T., and Krishna, N. R. (2000) Expression of 
functional recombinant beta-neurotoxin Css II in E. coli. Peptides 6, 767-772. 

4. Pierce, J. C. (1994) In-frame cloning of large synthetic genes using moderate-size oligo- 
nucleotides. BioTechniques 16, 708-715. 

5. Yanisch-Perron, C, Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vec- 
tors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 
33, 103-119. 

6. Barnett, R. W. and Erflel, H. (1990) Rapid generation of DNA fragments by PCR amplifi- 
cation of crude, synthetic oligonucleotides. Nucl. Acids Res. 18, 3094. 

7. Dillion, P. J. and Rosen, C. A. (1990) A rapid method for the construction of synthetic 
genes using the polymerase chain reaction. BioTechniques 9, 298-300. 

8. Sandhu, G. S., Aleff, R. A., and Kline, B. C. (1992) Dual asymmetric PCR: One-step 
construction of synthetic genes. BioTechniques 12, 14-16. 

9. Birnboim, H. C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening 
recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1523. 

10. Kane, J. F. (1995) Effects of rare codon clusters on high-level expression of heterologous 
proteins in Escherichia coli. Curr. Opin. Biotech. 5, 494-500. 

11. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory 
Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 



20 

Megaprimer PCR 

Sailen Barik 

1. Introduction 

A large variety of procedures of site-directed mutagenesis based on polymerase 
chain reaction (PCR) have been developed over the last decade. Among them, the 
"megaprimer" method, originally reported in 1990 (7), and its subsequent updates 
(8,10) still retain their popularity because they combine simplicity and versatility. Our 
most recent search in the PUBMED, using "megaprimer" as the keyword, generated 
24 publications, many of which were improvements on the original theme. This is an 
impressive number, considering that "megaprimer" is essentially a specialized tech- 
nique. In this chapter, we provide an updated protocol incorporating the variations and 
improvements of the basic technique published over the past decade. These include: a 
combination of magapriming and overlap extension, improvement of yield, use of 
single-stranded DNA, spiking with a proofreading polymerase (e.g., Pfu) to avoid 
unwanted mutations arising from nontemplated insertions by Taq polymerase, and 
the inclusion of various kinds of mutations, including multiple, nonadjacent ones 
(2-12,18,19,26-32). 

The basic method, described in Fig. 1, still requires three oligonucleotide primers 
and two PCRs (termed PCR-1 and -2 here) employing the wild-type DNA as template 
(1,2,8,10). The "mutant" primer is represented by M, and the two "flanking" primers, 
by A and B. The M primer may encode a substitution, deletion, insertion, or a combi- 
nation of these mutations, thus providing versatility while using the same fundamental 
strategy (10). The first PCR (PCR-1) is performed using the mutant primer M and one 
of the flanking primers, such as A (see Fig. 1). The double-stranded product A-M is 
purified and used as a primer (hence the name "megaprimer"; ref. /) in the second 
PCR (PCR-2) together with the other flanking primer B. Note that both strands of the 
megaprimer have the potential to prime on the respective complementary strands of 
the template. However, the fundamental principles of PCR amplification ensure that 
only that strand of the megaprimer, which extends to the other primer (B in Fig. 1), 
will be exponentially amplified into the double-stranded product in PCR-2. As afore- 
mentioned, the wild-type DNA is used as template in both PCRs. 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

189 



190 Bank 

A 
-— ► Wild type template 



I 



M 
PCR-1 



A-M 

• 



Mutant megaprimer 
A-M 




dfc 



Wild type template ^ B 
PCR-2 



I' 



Mutant product (A-M-B) 

: = 



Fig. 1. The basic megaprimer method. Primers A, B, M, and the priming strand of the 
megaprimer AM are indicated by thinner lines with arrowhead, while the thicker double lines 
represent the wild type template (usually part of a plasmid clone, not shown). Primers A and B 
contain restriction sites (e.g., Ndel andBamHI) indicated as thicker regions, and extra "clamp" 
sequence at the 5' end indicated by double lines. The sequence to be inserted is shown as the 
dotted region in primer M and the subsequent PCR products. The final product containing the 
insertion is restricted and cloned. 



Poor yields from PCR-2 have sometimes been reported, especially when the 
megaprimer is large (0.8 kb and above). Although the exact reason remains unclear, it 
must have something to do with the unique features of the megaprimer: its double- 
stranded nature and large size. Strand separation of the double-stranded megaprimer is 
essentially achieved in the denaturation steps of the PCR cycle. Under some condi- 
tions, however, self-annealing of the megaprimer apparently tends to reduce the yield 
of the product (4). 

Here, we provide a brief overview of the various solutions suggested to overcome 
the low yield. In one approach, a biotin tag is added to the 5' end of primer A, which 
generates a biotin-labeled megaprimer in PCR-1. The megaprimer is denatured, and 
the biotinylated strand is purified on streptavidin-coupled magnetic beads (30). In 
another method (31), the use of two parallel templates allowed inclusion of two flank- 
ing primers as well as the megaprimer in PCR-2, resulting in a direct amplification of 
the final product. Use of a "one-tube" method (see Note 6), when properly optimized, 
may help eliminate the loss of megaprimer during the purification step. Other strategies 
for increasing the yield of PCR-2 rely on optimizing the concentrations of template 
and megaprimer (4,11,12,32). The use of higher amounts of template (in the microgram 



Megaprimer PCR 1 9 1 

range, as opposed to nanogram quantities used in standard PCR) in PCR-2 has been 
shown to dramatically increase the product yield for some sequences (4). A more gen- 
eral strategy, however, is to increase the amount of the megaprimer. A method that we 
have found useful is to carry out the first several cycles of PCR-2 with the megaprimer 
only. After this initial asymmetric PCR, the small primer is added (11), and PCR is 
continued. In an optimized method (31), the starting concentration of megaprimer is 
increased to 6 pg (from 25 ng) per 100 pL PCR-2. We have adopted a combination of 
the last two findings in this article. 

2. Materials 

1. Template: About 100 ng DNA template to be mutated (e.g., a gene cloned in a plasmid). 

2. Primers: 100 pg of oligonucleotide primers A and B (see Fig. 1), and 50 ng of mutant 
primer M; one primer, say A, in the opposite sense, and other primer, B, in the same sense 
as the mutant primer M. If the plan is to digest the final product with restriction enzymes 
for the purposes of cloning, include restriction sites, preferably unique, in these primers. 
Realize that the mutant primer may contain a point mutation, or insertion, or deletion, as 
desired (see Notes 1 and 2). 

3. PCR buffer: 10X PCR buffer for Pfu polymerase (Stratagene Cloning Systems, La lolla, 
CA) is 200 mM Tris-HCl (pH 8.0-8.3), 100 mM KC1, 20 mM MgCl 2 , 60 mM ammonium 
sulfate, 1% Triton X-100, 100 pg/mL nuclease-free BSA. The buffer is usually supplied 
with the enzyme. 

4. Deoxyribonucleotides: Use a final dNTP concentration of 200 \iM for each nucleotide. 
Make a stock dNTP mix containing 2 mM of each dNTP (dATP, dCTP, dGTP, dTTP). 
We make it by adding 50 pL of 10 mM stock solutions of each nucleotide, available 
commercially, into 50 pL H 2 0, to produce 250 pL of the stock mix. 

5. Analysis and purification of DNA: A system for purifying the PCR products, such as gel 
electrophoresis, followed by recovery of the appropriate DNA band in the excised agar- 
ose fragment. 

Wherever needed in this procedure, use deionized (e.g., Millipore) autoclaved water. 

3. Methods 

3.1. PCR-1: Synthesis of the Megaprimer 

1. We have assumed that the reader is familiar with the basic PCR. Use the following recipe 
for the first PCR. Make the following 100 pL reaction mix in an appropriate micro- 
centrifuge tube (0.5 or 1.7 mL, dictated by the heating block of your thermal cycler): 

H 2 75 pL 

10X PCR buffer 10 pL 

dNTP mix (2 mM each) 10 pL 

(final concentration of each nucleotide is 200 pM) 
Primer A 50 pmole 

Primer M 50 pmole 

DNA template 1 0- 1 00 ng 

Pfu polymerase (2.5 U) 0.5 pL 

(or 2.5 U Taq plus 0.1 U Pfu polymerase, see Note 1) 
(Total = 100 pL) 

2. Vortex to mix, then spin briefly in a microfuge. If the thermal cycler has a heated lid, then 
proceed to perform PCR; otherwise, reopen the tube, overlay the reaction mixture with 



192 Bank 

enough mineral oil to cover the reaction (approx 100 uL for a 0.5-mL microfuge tube), 
then close cap. The tube is now ready for thermal cycling. 

3. Perform PCR-1 using the following cycle profiles. 

Initial denaturation 94°C, 3 min 

30-40 main cycles 94°C , 1 min (denaturation) 

T°C (depending on the T m of the primers), 2 min (annealing) 
72°C , appropriate time, depending on product length (extension) 
Final extension 72°C , 1.5X n min 

Following synthesis, the samples are maintained at 4°C (called "soak" file in older 
Perkin-Elmer programs) for a specified time. Some instruments lack an active cooling 
mechanism and keep samples at an ambient temperature of about 20°C by circulating tap 
water around the heat block, which appears to be adequate for overnight runs. 

4. After PCR, proceed directly to the next step if there is no oil overlay. Otherwise, first 
remove the oil as follows. (If oil is not removed completely, the sample will float up when 
loaded in horizontal agarose gels !) Add 200 uT of chloroform to each tube. The mineral 
oil and chloroform will mix to form a single phase and sink to the bottom of the tube. Spin 
for 30 s in a microfuge. Carefully collect approx 80 \iL of top aqueous layer and transfer 
to a fresh Eppendorf tube. 

5. Purify the megaprimer using any standard procedure of your choice (as long as the 
nonmutagenic primer A is removed) and use it in PCR-2 below. 

3.2. PCR-2: Synthesis of the Mutant Using the Megaprimer 

1. Reconstitute 100 [iL PCR as follows: 

10XPCR buffer 10 (iL 

dNTP mix (2 mM each) 10 ixL 

(final concentration of each nucleotide is 200 \iM) 
All of the recovered megaprimer (A-M) 
from the previous step (see Note 3) 20-50 u,L 

DNA template 0.2 \ig 

Make up volume to 100 \iL with H 2 0. 
Mix well. 

2. Start reaction essentially as described for PCR-1, except that a "hot-start" is preferred 
(see Note 4) and is performed as follows. When the reaction is in the annealing step of the 
first cycle, open the cap briefly, quickly add 0.5 [xL Pfu polymerase (2.5 U) (or 2.5 U Taq 
plus 0.1 U Pfu polymerase), and mix by pipeting. Close the cap and let PCR continue. 

3. After five cycles, when the reaction is again at an annealing step, promptly add 50 pmole 
of primer B, mix well, and let PCR continue another 30 cycles. (The small amounts of 
primer B and Pfu polymerase do not contribute significantly to the total reaction volume 
and therefore, have been ignored in the volume calculations.) 

4. Do another PCR in parallel, using primers A and B (and no megaprimer) and the same 
wild-type template; use an aliquot (5 u.L) of this PCR as a size marker when analyzing 
PCR-2 by gel electrophoresis. This will also help in identifying the real product (in 
PCR-2) among the wrong ones that sometimes result from mispriming. 

5. Gel-purify the final mutant PCR product essentially as described earlier for the purifica- 
tion of the megaprimer (see Notes 5 and 6). Now it is ready for restriction, cloning, 
sequencing etc. 



Megaprimer PCR 193 

4. Notes 

1. The problem of nontemplate nucleotides and its solution. Perhaps the most unique feature 
of the megaprimer method is that the product of one PCR becomes a primer in the next, 
which creates the following potential problem. Taq polymerase, due to its lack of proof- 
reading activity, tends to extend the product DNA beyond the template by adding one or 
two nontemplate residues, predominantly A's (14). When the product is used as a primer 
in the next round of PCR (PCR-2), these nontemplated A residues may not match with the 
template, and therefore, will either abrogate amplification (15-17) or produce an unde- 
sired A-substitution. A variety of solutions to this problem have been recommended 
(5,8,10,18). The first is to design the mutant primer such that there is at least one T resi- 
due beyond the 5' end of the primer sequence in the template. Thus, when the comple- 
mentary strand incorporates a nontemplated A at the 3' end, it will still be complementary 
to the other strand. If the template sequence does not permit this, a second solution, which 
we have recommended in this chapter, is to use a mixture of Taq and Pfu DNA poly- 
merases in 20:1 ratio in PCR-2 (3), or to use Pfu exclusively. The 3' exonuclease activity 
of Pfu should remove any mismatch at the 3' end of the megaprimer; however, this proof- 
reading ability also necessitates the addition of at least ten perfectly matched bases on 
both the 5' and 3' ends of the mutagenic primer (8,10,15,19). We have not actually tested 
other polymerases that are proficient in proofreading; but some of them might be used in 
lieu of Pfu. 

In addition to these unique considerations, the general rules of primer design, some of 
which are described below, should be followed. 

2. Length of the megaprimer. Try to avoid making megaprimers (A-M) that approach the 
size of the final, full-length product (gene) A-B (see Fig. 1). Briefly, if M is too close to 
B, it will make separation of AB and AM (unincorporated, left-over megaprimer) diffi- 
cult after PCR. When the mutation is to be created near B, one should make an M primer 
of the opposite polarity, and synthesize BM megaprimer (rather than AM), and then do 
PCR-2 with BM megaprimer and the A primer. When the mutation is at or very near the 
5' or 3' end of the gene (within 1-50 nucleotides), there is no need to use the megaprimer 
method; one can simply incorporate the mutation in either A or B primer and do a straight- 
forward PCR using A and B primers! For borderline situations, such as when the muta- 
tion is, for example, 120 nucleotides away from the 5' end of the gene, incorporation of 
the mutation in primer A may make the primer too big to synthesize; or else, it will make 
the megaprimer AM too short to purify away from primer B. In such a case, simply back 
up primer A a few hundred bases further upstream in order to make the AM megaprimer 
longer. In general, realize that primers A and B can be located virtually anywhere on 
either side of the mutant primer M, and therefore, try to utilize this flexibility as an advan- 
tage when designing these primers. 

3. Molar amount of megaprimer. Since the megaprimer is large, one needs to use a greater 
quantity of it to achieve the same number of moles as a smaller primer. Example: 50 pmoles 
of a 20 nt-long single-stranded primer will equal 0.3 u,g; however, 50 pmoles of a 500 nt- 
long double- stranded megaprimer will equal 6 u,g. A good yield and recovery of mega- 
primer is, therefore, important. If needed, do 2X 100 ^L PCRs to generate the megaprimer. 
There is no need to remove the template DNA after PCR-1, because the same DNA will 
be used as template in PCR-2. 



194 Barik 

4. "Hot start" PCR-2. The hot-start technique used in PCR-2 works just as well as the more 
expensive comMercial methods. Hot start tends to reduce false and nonspecific priming 
in PCR in general (39) and is particularly useful in PCR-2 of the megaprimer method (our 
unpublished observation). 

5. Poor yield of mutant. If the final yield is poor, the surest strategy is to amplify a portion of 
the gel-purified mutant product in a third PCR (PCR-3) using primers A and B and hot 
start. This may also be necessary if PCR-2 produces nonspecific products in addition to 
the specific one. Before PCR-3 is carried out, however, it is very important to ensure that 
the mutant product of PCR-2 is well separated from the wild-type template in the gel 
purification; otherwise, PCR-3 will amplify the wild-type DNA as well. The final gel- 
purified mutant DNA (from either PCR-2 or PCR-3) is ready for a variety of applications, 
as described later in brief. 

6. Single-tube methods. Recently, various investigators have reported successful modifica- 
tions of the megaprimer method in which the purification step is either simplified or not 
required (19,26-29). One involves cleavage of the template, coupled with enzymatic 
removal of PCR-1 primers, to ensure amplification of the correct product in PCR-2 (27). 
A second possibility is to exploit the unusually high T m of the megaprimer by designing a 
short, low T m flanking primer for PCR-1, and a long flanking primer for PCR-2. This 
enables the use of a higher T m for PCR-2 such that it will only allow annealing of the 
appropriate flanking primer (28). A third method uses a limiting amount of the first flank- 
ing primer, such that when the second flanking primer is added, the principle product will 
be the mutant DNA (19). Although we have not tested any of these modifications, the 
interested reader is advised to consult the original papers. 

Acknowledgment 

Research in the author's laboratory was supported in part by NIH Grant AI 45803 
and by a Burroughs Wellcome New Initiatives in Malaria Research Award (to S.B.). 

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17. Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C, and Sninsky, 
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20. Rychlik, W. (1993) Selection of primers for polymerase chain reaction. Meth. Mol. Biol. 
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25. Zintz, C. B. and Beebe, D. C. (1991) Rapid re-amplification of PCR products purified in 
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26. Ling, M., and Robinson, B. H. (1995) A one-step polymerase chain reaction site-directed 
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27. Seraphin, B. and Kandels-Lewis, S. (1996) An efficient PCR mutagenesis strategy with- 
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31. Upender, M., Raj, L., and Weir, M. (1995) Megaprimer method for in vitro mutagenesis 
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tion with transcript sequencing. Science 239, 491-494. 



21 

PCR-Mediated Recombination 

A General Method Applied 

to Construct Chimeric Molecular Clones 

Guowei Fang, Barbara Weiser, Aloise Visosky, Timothy Moran, 
and Harold Burger 

1. Introduction 

Molecular cloning has proven to be a powerful tool in biology, and chimeric clones 
are useful in a variety of fields including microbial pathogenesis and the development 
of vaccines. Chimeras can be created from DNA by using conventional cloning tech- 
niques, specifically restriction cleavage and DNA ligation. Such techniques, however, 
have limitations; most commonly, limitations result from the lack of restriction sites 
to provide points of entry for inserts in the desired regions or the multiplicity of 
restriction sites in other regions of the DNA. Because recombinant DNA molecules 
may be created during polymerase chain reaction (PCR) when two or more different 
DNA sequences are brought together (1,2), PCR-mediated recombination has been 
exploited to join DNA fragments of a few hundred bases (3-8). There are two draw- 
backs to these methods. First, they often involve multiple steps, and second, sequence 
errors frequently are introduced by certain thermostable polymerases during the PCR 
reaction (9,10). 

We have developed a widely applicable, improved method to construct recombi- 
nant DNA molecules without reliance on restriction sites. The method differs from 
older PCR-mediated recombination procedures (3-8) in several ways: it is useful for a 
wide range of constructions, ranging from a few hundred bases to approximately 10 kb; 
it is based on asymmetric PCR that greatly increases the yield of desired products; it 
employs high-fidelity DNA polymerase; and results in a very low error rate. The tech- 
nique utilizes PCR-amplified DNA, including DNA synthesized from RNA by reverse 
transcription. 



From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 



197 



198 Fangetal. 

To demonstrate the power of PCR-mediated recombination and provide an example 
of the general utility of the method, we constructed chimeric infectious molecular 
clones of HIV- 1 derived from plasma viral RNA and proviral long-terminal repeats 
(LTR)s. To study pathogenesis and develop vaccines, it would be desirable to use 
infectious HIV and SIV clones derived from plasma viral RNA, which is more repre- 
sentative of the replicating virus pool than proviral DNA (11,12). The PCR-based 
method described here made construction of such clones possible without reliance on 
restriction sites. DNA sequences were first amplified by high-fidelity PCR using Pfu 
polymerase; they were then used both as megaprimers and templates in subsequent 
asymmetric long PCR amplifications to form chimeric clones. Biological character- 
ization of these clones showed that most were infectious in tissue culture and sequence 
analysis demonstrated an error rate of only one base change in 20 kb of DNA sequence. 

2. Materials 

2. 1. Clones and Strains 

The sources of DNA are the following: plasmid HIV-1 pNL4-3 (13), which con- 
tains a full-length HIV-1 proviral genome; plasmid FG901-18, which contains the 5' 
half of an HIV-1 cDNA genome (5 kb); and plasmid FG902-12, which contains the 3' 
half of the same HIV-1 genome (5 kb). Both plasmids FG901-18 and FG902-12 are 
derived from plasma viral RNA from Wads worth Center patient 001 (WC1-PR) and 
were constructed as previously described (14). The Escherichia coli strain used for 
transformation is X-blue ultra (Stratagene, La Jolla, CA). 

2.2. PCR Primers 

A set of oligonucleotide primers are designed and synthesized to amplify particular 
DNA fragments. Each primer contains the sequences necessary to amplify the frag- 
ment of interest from the source DNA; it also contains the 3' and 5' sequences comple- 
mentary to the ends of the other DNA fragments chosen to be joined. Because the 
amplified fragments serve as megaprimers and templates in subsequent PCRs, the 
sequence and length of the overlap region is also critical. The primers are designed to 
achieve annealing at temperatures a60°C and to avoid hairpins, self-priming, and 
primer-dimer formation. Highly conserved sequences are selected as primer sites based 
on available HIV-1 sequence data. Primers designed for amplifying the 5' end of the 
LTR region of HIV-1 pNL4-3 (GenBank Ml 9921) are: NL5F (forward) 5'-TGG AAG 
GGC TAA TTT GGT CCC AAA AAA G-3'; NL5R (reverse) 5'-CAT CTC TCT CCT 
TCT AGC CTC C-3' . The primers for the 3' end of the LTR region of HIV-1 pNL4-3 
are: NL3F (forward) 5'-CAC AAG TAG CAA TAC AGC AGC TAC CAA TGC-3'; 
and NL3R (reverse) 5'-TGC TAG AGA TTT TCC ACA CTG AC-3'. 

Primers for amplifying the 5' half of the HIV-1 cDNA genome (5 kb) derived from 
the plasma viral RNA of patient WC1-PR are: FGF60 (forward) 5'-CAG ACC CTT 
TTA GTC AGT GTG GAA AAT C-3'; and FGR53 (reverse) 5'-GTC TAC TTG TGT 
GCT ATA TCT CTT TTT CCT CC-3'. The primers for amplifying the 3' half genome 
(5 kb) are: FGF46 (forward) 5'-GCA TTC CCT ACA ATC CCC AAA G-3'; and 
FGR95 (reverse) 5'-GGT CTA ACC AGA GAG ACC CAG TAC AG-3'. Primers were 
prescreened for optimal sensitivity and efficiency. 



PCR-Mediated Recombination 199 

2.3. Polymerase Chain Reaction (PCR) 

High-fidelity PCRs are performed using Pfu DNA polymerase (Stratagene). 
AmpliMax PCR® Gems (Perkin-Elmer, Foster City, CA) are used to minimize undes- 
ired primer interaction. Dpnl endonuclease (BioLabs, Beverly, MA) is added to the 
reaction to digest the parental DNA template after PCR. The PCR products are ana- 
lyzed by gel electrophoresis, and the desired fragments are isolated and purified using 
the QIAquick Gel Extraction kit (Qiagen, Chatsworth, CA). 

Long asymmetric PCR amplification and construction use both Tth XL (Perkin- 
Elmer) and Pfu polymerases. All of the reactions are run in a Perkin-Elmer GeneAmp 
9600 thermal cycler (Perkin Elmer). The PCR products are assayed on 0.8% SeaKem 
GTG agarose gel (FMC, Rockland, ME). 

2.4. Cloning and Colony Screening 

The purified DNA fragments are directly ligated into a phagemid TA vector (pCR 
II plasmid) by using the Original TA Cloning Kit (Invitrogen, Carlsbad, CA). The 
plasmid is used to transform X-blue ultra E.coli competent cells (Stratagene). After 
transformation, white colonies from X-Gal plates are verified by restriction digestion 
and partial DNA sequencing. 

2.5. DNA Transfection and Virus Replication Assays 

Plasmid DNA (10 u,g of each) encoding HIV-1 genomes are transfected into nor- 
mal human dermal fibroblast cell (NHDF 710, Clonetics, San Diego, CA) by the cal- 
cium phosphate precipitation method and Mammalian Transfection kit (Stratagene). 
Supernatant are harvested, clarified, and used to infect peripheral blood mononuclear 
cells (PBMCs) using standard methods. 

HIV-1 reverse transcriptase assays are performed using the Reverse Transcriptase 
Assay, Nonradioactive kit (Boehringer-Mannheim, Indianapolis, IN) following the 
manufacturer's instructions; HIV-1 p24 antigen capture assays are performed using 
the Alliance™ HIV-1 p24 ELISA kit (Dupont, Boston, MA). 

2.6. Sequence Analysis 

Plasmid DNA is extracted and purified using the Qiagen Max kit (Qiagen). The 
DNA templates are sequenced using fluorescent dye-labeled terminators and an 
Applied Biosystems DNA sequencer (Applied Biosystems, Foster City, CA). Sequence 
data are analyzed using University of Wisconsin Genetics Computer Group Software 
Packages on a Sun computer. The full sequences of two infectious HIV-1 clones, 
FG9012-38 and FG9012-40, have been contributed to GenBank under accession num- 
bers AF003887 and AF003888. 

3. Methods 

3.1 . Construction of Chimeric HIV-1 Clones 

To construct full-length infectious clones of HIV-1, we first used a method we 
previously developed to clone complete HIV-1 genomes directly from plasma viral 
RNA using long reverse transcription and PCR (RT-PCR) (14,15). Long cDNAs were 



200 Fang et al. 

derived from the HIV-1 RNA genome. Then, two 5-kb DNA fragments containing the 
5' and 3' half-genomes were amplified and cloned. Because of the complex replication 
cycle of retroviruses, the sequence of the virus that appears in the RNA form, the form 
found in virions, differs in the LTR region from that found in the DNA or proviral 
form, which is found integrated in cells. For this reason, the HIV-1 genome cloned 
from virion-associated RNA has incomplete LTRs; it needs to have complete LTRs 
attached for clones to be infectious as DNA. Therefore, DNA fragments from proviral 
5' and 3' LTR regions were linked to the 5' and 3' ends of the constructs, respectively. 
DNA fragments were obtained from two sources in the experiments described here: 
cloned cDNA reverse transcribed from plasma viral RNA (plasmids FG901-18 and 
FG902-12) and cloned proviral DNA (plasmid pNL4-3, which contains a full-length 
HIV-1 LTR). Construction of clones using conventional restriction and ligation meth- 
ods was not feasible in this case because of the absence of convenient restriction sites 
in these HIV-1 genomes. We, therefore, developed a strategy to assemble the full- 
length HIV-1 clones by using PCR-mediated recombination (see Fig. 1). 

3.2. High Fidelity Long PCR 

The four DNA fragments (A, B, C, D) encompassing the full-length HIV-1 genome 
were first amplified individually in a high fidelity PCR by using Pfu DNA polymerase. 
Each of the fragments shared a segment of an overlapping sequence with one or two 
other fragments. 

The optimal conditions for using Pfu polymerase in our high fidelity PCR were 
evaluated (see Notes 1 and 2). The final conditions for the amplification using 
Pfu polymerase were: 10 cycles of PCR at 94°C for 15 s, 55°C for 45 s, and 72°C for 
2-10 min in a final volume of 100 |iL with 0.2 u,g plasmid DNA template, 2.5 U of Pfu 
polymerase, 20 pmol of each primer, 20 \iM each dNTP, and 0.5X Pfu buffer (contain- 
ing 1.0mMMg 2+ ). 

Because of the high initial concentration of template used, to eliminate the carryover 
contamination of parental templates, the restriction endonuclease Dpnl was added to 
the reaction after PCR. Dpnl is specific for methylated and hemimethylated DNA; it 
was used to digest parental plasmid DNA and to select for amplified DNA. 

Using the optimized PCR conditions, four fragments were amplified from corre- 
sponding plasmids. They are: fragment A, a 792-bp fragment amplified from HIV-1 
pNL4-3, which contains the entire 5'LTR; fragment B, a 5-kb 5' half genome from 
plasmid clone FG901-18; fragment C, a 5 kb 3' half genome from plasmid clone 
FG902-12 and fragment D, a 750 bp fragment from 3' end of HIV-1 pNL4-3, which 
contains the entire 3' LTR (see Fig. 2). All four fragments to be assembled were gel 
purified and quantitated individually. 

3.3. Asymmetric PCR Recombination 

After the first PCR, an asymmetric PCR, which favors single-strand extension in 
the desired direction, was used in order to construct the full-length molecule (see 
Fig. 1). A combination of Pfu and rTth polymerase was used in the asymmetric PCR 
amplification and construction to take advantage of the Pfu polymerase's precision as 
well as to reduce PCR time, increase PCR yield, and enable us to use TA vector clon- 



PCR-Mediated Recombination 



201 



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Fig. 1. Schematic diagram of the PCR-mediated recombination strategy used in construct- 
ing HIV-1 chimeric infectious clones. The segments of the HIV-1 genome in plasmid vectors 
are shown as rectangles with the plasmid name on the top; each plasmid is color coded. Syn- 
thetic oligonucleotide primers are shown as single strands, with full arrowheads indicating the 
direction. The PCR-amplified products are shown as two paired strands and are named by capi- 
tal letters A-D and color coded according to the plasmid template. A and D are both derived 
from plasmid pNL4-3 (15) and, therefore, are depicted in one color. The intermediate PCR 
products are shown as single strands, with half arrowheads indicating the direction in which 
each strand can act as a primer for DNA polymerase (the 5'-3' direction). 



202 Fang et al. 

M A ■ C D 11 VI 




Fig. 2. Agarose gel analysis of DNA fragments amplified by PCR. Lane A shows a 792-bp 
fragment amplified from HIV-1 pNL4-3, which contains the entire 5' LTR; lane B, a 5 kb 5' 
half genome from plasmid clone FG901-12; fragment C, a 5-kb 3' half genome from plasmid 
clone FG902-12; and fragment D, a 750 bp fragment from the 3' end of HIV-1 pNL4-3, which 
contains the entire 3' LTR. Lane FL shows the full-length 10 kb HIV-1 fragment constructed by 
PCR-mediated recombination. Lane M depicts a set of molecular weight standards (1 kb lad- 
der; GIBCO BRL/Life Technologies). Numbers on the left indicate the molecular weight (bp) 
of some of the bands in lane M. The source of the plasmids is given in the text. 



ing. The four PCR-amplified fragments (A, B, C, and D) were mixed in an asymmetric 
ratio with two synthetic oligonucleotide primers, which serve as internal primers. Frag- 
ments A and D were added in excess and served as external flanking megaprimers, 
whereas the quantity of the two internal oligonucleotide primers was very limited. 
Conditions for asymmetric PCR were evaluated (see Notes 2 and 3). The optimal con- 
ditions were: 2 jxg of each external flanking fragments (A and D, megaprimers), 0.1 jj,g 
of each 5-kb internal fragment (B and C) produced by high fidelity PCR were mixed 
with 0.2 pmol of synthetic oligonucleotide primers (FGR53 and FGF46). The mixture 
was subjected to 20 cycles of PCR in a volume of 100 \iL containing 2.5 U of rTth 
polymerase, XL, 0.004 U of Pfu polymerase, 10 \iM each of dNTPs, 1.2 mM 
Mg(OAc) 2 , and IX XL PCR buffer II. The PCR cycling parameters were 94°C for 
15 s, and 72°C for 8 min. 

The resulting PCR produced an asymmetric single-strand amplification of two 5-kb 
halves (AB and CD) of the genome as the majority forms. In the subsequent PCR 
cycles, these intermediate single-stranded DNAs, each representing half of the genome 
(AB and CD) overlapped and annealed to complementary strands of each other. The 
annealed strands then served as megaprimers for one another and were extended by 
PCR to form full-length molecules (see Fig. 2). It was not necessary to purify the 
intermediate products. As soon as the first 10-kb full-length strand was synthesized, it 
too was PCR-amplified using the flanking primers A and D. 

The 10-kb PCR-amplified fragment was detected by gel electrophoresis as a major 
band (see Fig. 2). The correctly assembled DNA was a major species and could be 
easily separated by electrophoresis from the portions of the genome serving as paren- 
tal templates. The full-length product was then isolated and directly cloned into a TA 



PCR-Mediated Recombination 203 

i 

A 

Vnlur 

C— 

■ — 



i i c i i i : i x i <j ■ 

-1 - * * a * c , i _ 




F u t bm ifc chloric rilMt I*** 

__ j J: t a t £_j _ 



fWAWM it™ 

Fig. 3. Restriction digestion of constructed full-length HIV-1 molecular clones. Lanes 1 and 
2 are two full-length chimeric HIV-1 molecules constructed by PCR-mediated recombination, 
with intact LTRs, and cloned into the TA vector (Invitrogen). Lane 3 is a full-length cDNA 
clone generated directly from plasma HIV-1 RNA as previously described (13) and cloned in 
the TA vector; it has partial LTRs. Plasmid DNA was digested with EcoRI. The bottom part of 
the figure is a schematic diagram of EcoRI restriction sites (shown as arrows) on cloned HIV-1 
genomes (shown as rectangles) and vectors (shown as single strands); the shadowed parts are 
HIV-1 LTR regions. Lane M depicts a molecular weight standard (1 kb ladder; Gibco-BRL/ 
Life Technologies). 



vector. The structure of the chimeric DNA product was verified by restriction diges- 
tion patterns (see Fig. 3) and partial DNA sequencing. Most of the clones screened 
contained complete or nearly complete HIV-1 genomes with the proper length and 
organization; no unwanted deletions or insertions in overlap regions were found. 

3.4. Sequence Fidelity of Clones 

To determine the sequence fidelity of full-length clones constructed by PCR-medi- 
ated recombination, two of the constructed full-length infectious clones (clones 
FG9012-38 and FG9012-40, GenBank accession numbers AF003887 and AF003888) 
and their parental plasmids were entirely sequenced. Only one mutation was found in 
clone 38 and none in clone 40. The PCR-introduced error was a transition (A to G), 
and was located in the untranslated region. No other mutations including frame shifts, 
deletions, or insertions were found. 

The cumulative error rate after PCR amplification of two complete genomes was 
0.005%, or a total of one base change detected in greater than 20 kb of plasmid DNA. 
This error rate is much lower than the previously reported rates of 0. 14-0.35% obtained 
by using thermostable enzymes (9,10,16). The high precision in the data reported here 
most likely resulted from using high fidelity polymerase and controlled PCR condi- 
tions aimed at producing a low error rate (see Notes 2 and 4). 



204 Fang et al. 

3.5. Infectivity of Constructed Clones 

The full-length chimeric molecules constructed from plasma-derived HIV-1 RNA 
and proviral DNA-derived LTRs were tested for infectivity by transfection into human 
dermal fibroblast cells. Controls included the transfection of pNL4-3 plasmid DNA 
and the TA vector plasmid DNA. Approximately half of the constructed clones tested 
(5 of 9 clones) produced virus particles as determined by both HIV-1 p24 antigen and 
reverse transcriptase assays. These particles were confirmed to be infectious virus by 
performing either cocultivation (14) of the transfected cells with PBMCs or by inocu- 
lation of donor PBMCs with cell-free supernatant from transfected cells. No virus was 
detected from the control experiments using vector plasmid DNA only. 

4. Notes 

1. The optimal concentration ranges of dNTPs (10-100 \iM each dNTP), DNA templates 
(50-2000 ng per reaction), PCR extension times (0.5-3 min per kb DNA to be amplified) 
and numbers of PCR cycles (5-25 cycles) were evaluated (data not shown). The final 
conditions for the amplification using Pfu polymerase were: 10 cycles of PCR at 94°C for 
15 s, 55°C for 45 s, and 72°C for 2-10 min in a final volume of 100 [xL with 0.2 u.g 
plasmid DNA template, 2.5 U of Pfu polymerase, 20 pmol of each primer, 20 \iM each 
dNTP, and 0.5X Pfu buffer (containing 1.0 mM Mg 2+ ). To construct infectious molecular 
clones using PCR-mediated recombination, the PCR polymerase used needs to have the 
lowest error rate possible to minimize the chance of producing unwanted mutations. Pfu 
polymerase has the lowest error rate of all known thermophilic polymerases (17,18). 
Unlike some polymerases (e.g., Taq, Tth), Pfu produces perfectly blunt ends of PCR 
products. The blunt end avoids the introduction of undesired adenosine residues onto the 
3' ends of the PCR products, which may lead to insertion of an extra nucleotide onto the 
full-length molecule during subsequent PCR. 

2. As an additional consideration, the PCR error rate is related to the number of PCR cycles. 
To minimize the number of PCR cycles for DNA synthesis, the highest concentration of 
template plasmid consistent with amplification should be employed. Because PCR error 
rates increase with the Mg 2+ and dNTP concentrations (17), the lowest Mg 2+ and dNTP 
concentrations compatible with amplification should be used. Conditions for asymmetric 
PCR, minimum Mg 2+ requirement, ratios of each fragment and primers, and the neces- 
sary length of overlap were investigated to develop the optimal conditions. 

3. The length of the overlap sequence influences the efficiency of PCR and its yield. 
Therefore, it is important to define the required length of overlap, particularly when the 
overlap region between two fragments includes basepairs that are not identical. To deter- 
mine the minimum length of overlap required for construction of 10-kb clones, we tested 
overlapping lengths ranging from 50 bp up to 690 bp, between fragments B and C, which 
have almost identical overlapping sequences (99.43% similarity). Because an overlap of 
270 bp between B and C did produce 10-kb products, longer overlaps (560, 640, and 
690 bp) resulted in a higher yield of full-length molecules. To increase the yield of 
complete products in PCR with shorter overlaps, more PCR cycles are needed, which 
may result in more errors being introduced. An overlap region of 50-100 bp per kb of 
DNA to be assembled appeared adequate for most long DNA constructions (data not 
shown). For overlapping regions with heterogeneous sequences, the optimal length of 
overlap needs to be determined and a longer overlapping sequence may be required. 



PCR-Mediated Recombination 205 

In the overlap regions between fragments A and B, and C and D, there were 3.66 and 
6.42% nucleotide differences respectively, yet fragments could still be assembled by using 
the relatively long overlapping (180 and 590 bp). 

The effect of the concentration of megaprimers on production of complete clones was 
studied by using from 0.2 u,g up to 2 u.g of megaprimers (DNA fragments A and D) per 
0.1 u.g of templates (DNA fragments B and C) in PCRs. The production of 10-kb product 
was correlated with the concentration of megaprimers; high concentration of megaprimers 
greatly improved the yield of final products (data not shown). A high concentration of 
megaprimers may facilitate the formation of intermediate strands AB and CD in asym- 
metric PCR amplification and increase the yield of final products. 

Because there were nucleotide differences between fragments A and B, and C and 
D, it was possible to determine which parental templates were included in the final full- 
length molecule. The sequence analysis indicated that the overlapping sequences 
between fragments A and B; and C and D in the full-length molecule were derived 
from HIV-1 pNL4-3 (A and D). This result confirmed our hypothesis that, in most 
cases, the two 5-kb halves (AB and CD) of the genome were first synthesized as single- 
stranded intermediates that then formed a full-length molecule in subsequent PCR 
cycles (see Fig. 1). 
4. PCR-mediated recombination provides a powerful method of recombining DNA 
sequences from any source without reliance on restriction sites. What is required to per- 
form PCR-mediated recombination is the sequence of the 3' and 5' overlapping regions of 
the desired PCR products. This method may be extended to include construction of chi- 
meras between any DNA fragments lacking sequence homology. Such chimeras may be 
constructed by introducing overlapping sequences to one of the fragments (19). To 
ensure that unwanted mutations have not been introduced into the clones constructed by 
this method, each clone should be sequenced. Our results demonstrate that by using a 
high-fidelity polymerase and highly controlled PCR conditions, the PCR-introduced 
error rate can be greatly minimized. 

This new procedure may be used to construct infectious chimeras of HIV or SIV for 
studies of vaccines and pathogenesis. Moreover, the method is designed to exchange viral 
genes at precise boundaries to study individual gene products from different HIV 
genomes. It can also be used to construct expression vectors for production of specific 
proteins or delivery vectors for gene transfer and gene therapy. Finally, the technique 
described here provides a versatile tool to transfer genes or gene fragments from different 
sources for genetic investigation and engineering. 

Acknowledgments 

This work was originally published by Fang et al. (1999) in Nature Medicine (20). 
The authors thank Ellen Shippey and Anne Klugo for help in preparing the manuscript 
and the Wadsworth Center Molecular Genetics Core Laboratory for oligonucleotide 
synthesis for PCR. This work was supported in part by grants from the National Insti- 
tutes of Health (R01A133334 and U01AI35004 from the National Institute of Allergy 
and Infectious Disease and the National Institute on Drug Abuse). 

References 

1. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., et al. 
(1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA poly- 
merase. Science 239, 487-491. 



206 Fang et al. 

2. Meyerhans, A., Vartanian, J. P., and Wain-Hobson, S. (1990) DNA recombination during 
PCR. Nucl. Acids Res. 18, 1687-1691. 

3. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J., and Pease, L. R. (1989) Engineering 
hybrid gene without the use of restriction enzymes: gene splicing by overlap extension. 
Gene 77, 61-68. 

4. Horton, R. M., Cai, Z. L., Ho, S. N., and Pease, L. R. (1990) Gene splicing by overlap 
extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8, 
528-535. 

5. Yolov, A. A. and Shabarova, Z. A. (1990) Constructing DNA by polymerase recombina- 
tion. Nucl. Acids Res. 18, 3483-3486. 

6. Klug, J., Wolf, M., and Beato, M. (1991) Creating chimeric molecules by PCR directed 
homologous DNA recombination. Nucl. Acids Res. 19, 2793. 

7. Gu, H., Planas, J., Gomez, R., and Wilson, D. J. (1991) Full length mouse glycoghorin 
gene constructed using recombinant polymerase chain reaction. Biochem. Biophy. Res. 
Commun. Ill, 202-208. 

8. Sandhu, G. S., Aleff, R. A., and Kline, B. C. (1992) Dual asymmetric PCR: one-step con- 
struction of synthetic genes. Biotechniques 12, 14-16. 

9. Eckert, K. A. and Kunkel, T. A. (1991) DNA polymerase fidelity and the polymerase 
chain reaction. PCR Meth. Appl. 1, 17-24. 

10. Mattila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991) Fidelity of DNA synthesis 
by the Thermococcus litoralis DNA polymerase — an extremely heat stable enzyme with 
proofreading activity. Nucl. Acids Res. 19, 4967-4973. 

11. Coffin, J. M. (1995) HIV population dynamics in vivo: implications for genetic variation, 
pathogenesis, and therapy. Science 267, 483-489. 

12. Chun, T. W., Carruth, L., Finzi, D., Shen, X., DiGiuseppe, J. A., Taylor, H., et al. (1997) 
Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. 
Nature 387, 183-188. 

13. Myers, G. Korber, B., Foley, B., Jeang, K. T., Mellors, J. W., and Wain-Hobson, S. (1996) 
Human Retroviruses and AIDS. Los Alamos National Laboratory, Los Alamos, NM. 

14. Fang, G., Weiser, B., Visosky, A., Townsend, L., and Burger, H. (1996) Molecular clon- 
ing of full-length HIV-1 genomes directly from plasma viral RNA. ./. AIDS 12, 352-357. 

15. Fang, G., Burger, H., Grimson, R., Tropper, P., Nachman, S., Mayers, D., et al. (1995) 
Maternal plasma human immunodeficiency virus type 1 RNA level: a determinant and 
projected threshold for mother-to-child transmission. Proc. Natl. Acad. Sci. USA 92, 
12,100-12,104 

16. Salminen, M. O., Koch, C, Sanders-Buell, E., Ehrenberg, P. K., Michael, N. L., Carr, J. K., 
et al. (1995) Recovery of virtually full-length HIV-1 provirus of diverse subtypes from 
primary virus cultures using the polymerase chain reaction. Virology 213, 80-86. 

17. Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., and Mathur, 
E. J. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated 
from Pyrococcus furiosus. Gene 108, 1-6. 

18. Flaman, J. M., Frebourg, T., Moreau, V., Charbonnier, F., Martin, C, Ishioka, C, et al. 
(1994) A rapid PCR fidelity assay. Nucl. Acids Res. 22, 3259-3260. 

19. Horton, R. M. (1995) SOEing together tailor-made genes. Mol. Biotech. 3, 93-99. 

20. Fang, G., Weiser, B., Visosky, A., Moran, T., and Burger, H. (1999) PCR-mediated recom- 
bination: a general method applied to construct chimeric infectious molecular clones of 
plasma-derived HIV-1 RNA. Nat Med. 5, 239-242. 



22 



PCR Method for Generating Multiple Mutations 
at Adjacent Sites 

Jiri Adamec 
1. Introduction 

Site-directed mutagenesis is a commonly used tool for identifying the role of spe- 
cific amino acids in the structure and function of proteins. Various methods of in vitro 
mutagenesis have been described and are widely used for introducing modified coding 
sequences (1-7). In comparison, polymerase chain reaction (PCR)-based methods (1-6) 
are generally faster and more efficient than non-PCR-based methods (7). On the other 
hand, some PCR-based methods require two or more primers for each round of muta- 
genesis, whereas others need a single very long oligonucleotide or two round of PCR 
for introducing one mutation (5-7). These all can increase the cost of mutagenesis. 

The PCR method for generating multiple mutations at adjacent sites is a two-step 
procedure. This can be very efficient and economical in those cases where a large 
number of nucleotide (amino acid) changes, deletions or insertions are to be pro- 
grammed in a small region of the sequence. In the first step, a new, unique restriction 
site is introduced at the middle of or near to the part of DNA sequence to be changed 
without resulting in a change of the amino acid sequence. For this step, two PCR 
products cloned into a plasmid are used (see Fig. 1). The unique restriction site (URS3) 
is introduced into the plasmid by using overlapping oligonucleotide primers (P3 and 
P4) which contain sequences that complement the template DNA sequence at the 3'- 
end and each other at the 5'-end (see Fig. 1A). The sequences at the 5'-end of the 
primers contain the unique restriction site, which maintains the amino acid sequence 
of the expressed protein. Two PCR products are generated by using these internal 
overlapping primers (P3 and P4) paired with the corresponding external primers (PI 
and P2). The external primers PI and P2 cross the natural, unique restriction sites 
(URS 1 and URS2) of the plasmid (see Fig 1 A). The product of PCR I or PCR II is then 
cleaved with restriction enzymes corresponding to URS1 and URS3 or URS2 and 
URS3, respectively. Digested fragments are separated on agarose gel electrophoresis, 



From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 



207 



208 

A 



Adamec 




► P4 



P3<- 



URS31 



PCRI 



I 
URS3 

URS3 



PCRII 



URS2 



URS1 
1= 



Cleave by 
RE1 and RE3 



URS3 Cleave by 

RE3 and RE2 
URS3 



URS2 



=1 



URS3 



URS2 



Ligate into the plasmid 

previously cleaved by RE1 and RE2 




URS1 



URS3 



URS2 



Fig. 1. Principle of PCR method for generating multiple mutations at adjacent sites. Oligo- 
nucleotide primers are represented by the arrows at their annealing sites in the target DNA 
sequences of plasmid. (A) The two internal primers P3 and P4 containing the unique restriction 
site URS3 anneal to opposite strands of the DNA template at the region to be modified. The 
external primers PI and P2 cross the natural unique restriction sites URS1 and URS2, 
respectively. PCR I and PCR II are performed separately, their product cleaved by restriction 
enzymes (RE) corresponding to URS1 (RE1) and URS3 (RE3) or URS2 (RE2) and URS3 



Multiple Mutations at Adjacent Sites 
B 



209 




URSl 



URS3 



URS2 



PI 



PCR 



URSl 
i 



Ml or 
M2or 
M3 



M4or 
M5or 
M6 



-*> 



URS3 



PCR 



Cleave by RE1 and RE3 and 
ligate into the plasmid previously 
cleaved by RE1 and RE3 



URS2 



Cleave by RE2 and RE3 and 
ligate into the plasmid previously 
cleaved by RE2 and RE3 









URS3 



URS3 



URS3 



URS3 



URS3 



URS3 



Plasmids with mutated sequence 

Fig. 1. (continued from opposite page) (RE3), respectively, and the fragments are ligated into 
the plasmid previously cleaved by RE1 and RE2 enzymes, to create new plasmid containing 
URS3. (B) To generate mutations, mutagenic primers containing URS3 are paired with external 
primers PI (M1-M3, for generation of mutations at left side from URS3) or P2 (M4-M6, for 
generation of mutations at right side from URS3). The PCR fragments are then cleaved by 
appropriate restriction enzymes and used to reconstruct mutant sequences. Nucleotide changes 
are indicated as "•." 



210 Adamec 

extracted from gel slices, and ligated together into the original plasmid previously 
digested with restriction enzymes corresponding to URS1 and URS2 (see Fig. 1A). 
After transformation of Escherichia coli with ligated material, colonies are selected 
on LB plates (1% bacto tryptone, 0.5% bacto yeast extract, 1% NaCl and 1.2% agar) 
containing appropriate antibiotics. Plasmids are then isolated and tested for the 
presence of the URS3 site using a corresponding restriction enzyme, and those 
containing the URS3 site are used for the second step, which is the introduction of 
desired mutations. 

In the second step, mutagenic primers are synthesized to contain a URS3 sequence 
at the 5'-end and a sequence introducing a mutation at the 3'-end (see Fig. IB). In 
PCRs, mutagenic primers are paired with PI primer (primers M1-M3) or with P2 
primer (M4-M6), respectively (see Fig. IB). Amplified fragments, designated here as 
F1-F6, are digested by restriction enzymes corresponding to URS1 and URS3 (F1-F3) 
or by the enzymes corresponding to URS2 and URS3 (F4-F6), respectively. They are 
subsequently separated on an agarose gel, isolated, and individually ligated into the 
plasmid from first step previously digested with enzymes corresponding URS1 and 
URS3 for fragments F1-F3 or URS2 and URS4 for F4-F6, respectively (see Fig. IB). 
After transformation, isolated plasmids are tested for the presence of the correct muta- 
tion by sequencing. 

Using this approach, we were able to generate a large number of mutations to 
investigate the function of the oc-subunit of mitochondrial processing peptidase (8,9). 
Moreover, the very high efficiency of introducing mutations (90-95%) greatly reduced 
the number of colonies that needed to be screened, since we only had to test 2-3 
colonies for each mutation. 

2. Materials 

1. PCR machine. 

2. Plasmid containing DNA sequence to be mutated. 

3. Oligonucleotide primers. 

4. High-fidelity Pfu DNA polymerase and 10X Reaction Buffer (Stratagene, La Jolla, CA). 

5. PCR Nucleotide Mix (Promega, Madison, WI). 

6. QIAquick PCR Purification Kit (Qiagen, Valencia, CA). 

7. Low-melting agarose (e.g., SeaPlaque GTG agarose, FMC BioProducts, Rockland, ME). 

8. Reagents and apparatus for agarose gel electrophoresis. 

9. AgaxACE enzyme (Promega, Madison, WI). 

10. Restriction enzymes and buffers (e.g., from New England Biolabs, Beverly, MA). 

11. DNA ligase and buffer (e.g., from New England Biolabs). 

12. E. coli strain for transformation (e.g., MAX Efficiency DH5a Competent Cells, Life 
Technologies, Grand Island, NY). 

13. LB medium and LB agar (BIO 101, Vista, CA). 

14. Antibiotic(s) (e.g., from Life Technologies). 

15. QIAprep Spin Miniprep Kit (Qiagen). 



Multiple Mutations at Adjacent Sites 21 1 

3. Methods 

3.1 . Introduction of Unique Restriction Site 

3.1.1. Primer Design 

Primers PI and P2 should be approx 20 nt (nucleotides) in length with the sequence 
of the natural unique restriction site (URS) located in the middle. The choice of URS 
depends on the plasmid used. The ideal distance between primer PI and P3 or P2 and 
P4 is about 150 bps (basepairs). The primers P3 and P4, which introduce the new URS 
into the sequence at 5'-end, must be followed by certain number of any nucleotides. 
The number of these nucleotides varies and depends on the URS used as the restriction 
enzyme, because recognizing this site requires nucleotide sequence on both sites of 
URS to be efficient (see Note 1). In this process a critical factor is the selection of a 
new URS. There are four main criteria, namely: 

1 . the site is not present in the original plasmid sequence; 

2. the site is about the middle of region to be mutated; 

3. nucleotide changes do not affect amino acid composition; 

4. the restriction enzyme recognizing this site is commercially available. 

Hence, in order to fulfill this strategy, we generate a silent mutation as previously 
described (9) and highlighted in Fig. 2A. To determine possible silent mutations that 
can be introduced into the region of interest, we recommend using computer programs 
available on the Internet (e.g., WebCutter at http://www.firstmarket.com/cutter/ 
cut2.html). 

3.1.2. PCR, purification, and cloning 

1. Assemble two 50 uL PCRs (see Note 2): 

PCR I PCR II 

Template DNA (50 ng/uL) 2 uL 2 uL 

10X Reaction Buffer 5 u,L 5 u,L 

10X dNTP (2.5 mM each) 5 U.L 5 uL 

Primer PI (20 u.M) 1 u,L 

Primer P2 (20 \iM) — 1 uL 

Primer P3 (20 \iM) 1 \xL — 

Primer P4 (20 u.M) — 1 uL 

H 2 35 uL 35 uL 

Pfu DNA polymerase 1 uL 1 uU 

2. Use following steps to perform PCR: 

a. 94°C-5 min (initial denaturation); 

b. 94°C-30 s (denaturation); 

c. 50°C-30 s (annealing); 

d. 72°C-1 kb/min (extension); 

e. Repeat 30 times steps 2-4 (30 cycles); 

f. 72°C-10 min (final extension step). 



CQTSRDTTM (X)22 
-TGCCAGACCTCGAGAGACACCACCATG (N) 66 



-CCAGACCTCGAGAGACACCAC 



Xhol 



-*• PI 



(X)22 GLHRFCPVE 

( N ) 6 6 GGCCTGCACCGGTTTTGTCCTGTGGAG - 3 ' 



GGACG TGGCCA AAACAGGACA- 5 ' 
Agel 



P2 



IV) 

—0. 

rv> 



HPRLTDEEIEMTRMAVQFELEDLNM 
CACCCCCGCCTGACAGATGAGGAAATTGAGATGACGAGGATGGCTGTTCAGTTTGAACTTGAGGACCTGAACATG 

Find restriction sites which can be introduced by silent mutation. 
y Use Webcutter at "http: //www. firstmarket . com/ cutter / cut2 . html ". 



CCGCGG 



ACGCGT TGTACA 



Cfr42I Mlul 

Select restriction site. 

ACGCGT 
Design primers. 



BsrGI 



GAGCTC 
SacI 



1 



Mlul 



TAACTCTAC TGCGCA TACCGA- 5 ' 
P3 4 

5 ' -GAGATGACGCGTATGGCTGTT 



P4 



3 

CD 
O 



B 



CQTSRDTTM (X)22 
' -TGCCAGACCTCGAGAGACACCACCATG (N) 66 

5 ' -CCAGACCTCGAGAGACACCAC 



Xhol 



PI 



(X)22 GLHRFCPVE 

(N) 66 GGCCTGCACCGGTTTTGTCCTGTGGAG-3 ' 



P2 



GGACG TGGCCA AAACAGGACA- 5 ' 
Age! 



HPRLTDEEIEMTRMAVQFELEDLNM 
CACCCCCGCCTGACAGATGAGGAAATTGAGATGACGCGTATGGCTGTTCAGTTTGAACTTGAGGACCTGAACATG 



Mlul 



Design mutagenic primers. 



GGGGCGGACTGTCTTCTCCTTTAACTCTACTGCGCATAC - 5 ' 



GACTGTCTACTACTTTAACTCTACTGCGCATAC - 5 ' 



Ml (D->E) 



M2 (E-»D) 



ctT 

c 

sr 
^* 

o' 

CO 

to 

0)' 

O 
CD 

CO 

s-' 

CO 



TGTCTACTCCTATAACTCTACTGCGCATAC - 5 ' 



M3 (E->D) 



M4 (E->D) 5'- ATGACGCGTATGGCTGTTCAGTTTGATCTTGAGGAC 



M5 (E->D) 5'- ATGACGCGTATGGCTGTTCAGTTTGAACTTGATGACCTGAAC 



M6 (D-»E) 



5 ' - ATGACGCGTATGGCTGTTCAGTTTGAACTTGAGGAACTGAACATG 



Fig. 2. Primer design strategy for (A) introducing new, unique restriction site and (B) generating mutations (modified from ref. 9). Under- 
lined sequences indicate restriction enzyme sites. Bold letters indicate point mutations. 



—A 

CO 



214 Adamec 

3. Clean both PCR products using QIAquick PCR Purification Kit and protocols supplied 
by vendor. Use 50 uL of H 2 to elute DNA from cartridges. 

4. Cleave products of PCR I and PCR II, and plasmid with corresponding restriction enzymes 
(see Note 3). 

5. Purify digested PCR products and plasmid (see Note 4). 

6. Ligate purified PCR fragments and plasmid DNA from previous step. For ligation, use 
the molar ratio 3 : 3 : 1 of PCR I fragment : PCR II fragment : plasmid. Calculate DNA 
concentration of samples using: C (pmol/fiL) = (75 x A 260 x d) / (bp x 1), where A 260 is 
absorbance at 260 nm, d is dilution factor, bp is length of DNA in basepair, and / is cuvet 
width in cm. For ligation, add the following components to a microcentrifuge tube: 
cleaved PCR I fragment, PCR II fragment, plasmid, 2 uL 10X ligation buffer, 1 uL DNA 
ligase and H 2 to 20 uL. Incubate reaction overnight at room temperature. 

7. Transform appropriate E. coli strain using the following protocol (see Note 5). Mix liga- 
tion reaction from previous step with 100 uL of competent cells and keep on ice for 
30 min. Incubate the mixture at 42°C for 1 min, transfer back on ice for 1 min and then 
add 0.5 mL LB medium. Place on shaker and incubate an additional 1 h at 37 C C. Spin 
cells down (20 s at maximum speed), discard supernatant and resuspend cells gently 
in 200 uL LB medium. Plate on LB agar with appropriate antibiotic and let cells grow 
overnight at 37°C. 

8. Place individual colonies in 4 mL LB medium containing appropriate antibiotic and grow 
overnight at 37°C. Isolate plasmids using a QIAprep Spin Miniprep Kit following 
manufacture's protocol and cleave them with restriction enzyme corresponding to intro- 
duced restriction site URS3. Sequence positive plasmids (colonies that are cleaved with 
enzyme) and use for next steps. 

3.2. Mutations Generation 

1. To design mutagenic primers follow the protocols outlined in Subheading 3.1.1. 

2. For introduction of mutations on the left side from URS3, use the set up and conditions 
for PCR I described in steps 1 and 2 except the mutagenic primer is used instead of 
primer P3. Similarly, to introduce mutations on the right side from URS3 use set up and 
conditions for PCR II, but use the mutagenic primer instead of primer P4 (see Fig. 2B). 

3. Use the same protocol for purification, cleavage, and ligation as described in steps 3-6. 
Digested PCR fragments are then individually cloned into the cleaved plasmid, obtained 
from the previous step (for introduction of mutations on left side of URS3 use plasmid 
digested with restriction enzymes corresponding to URS1 and URS3, for mutations on 
right side, plasmid digested with restriction enzymes corresponding to URS2 and URS3). 
In the ligation reaction, combine the PCR fragment:plasmid in 3: 1 molar ratio. 

4. After transformation, plate cells on LB agar with appropriate antibiotic and let cells grow 
at 37°C overnight. Place two individual colonies from each transformation into 4 mL LB 
medium containing appropriate antibiotic and grow overnight at 37°C. 

5. Isolate plasmids using QIAprep Spin Miniprep Kit and sequence cloned fragments to 
confirm the presence of the mutations and to verify that no undesired changes occurred in 
the sequence during the PCR process. 

4. Notes 

1 . More detailed information about the cleavage close to the end of DNA fragments could 
be found in (10). Usually, it is enough to use 6 nt (e.g., AATAAT). 



Multiple Mutations at Adjacent Sites 215 

2. To decrease the number of undesired mutations generated by PCR process, we have used 
high-fidelity Pfu DNA polymerase and higher concentrations of template DNA (approx 
100 ng). If the PCR machine is without a heated lid, we used 50 (xL of mineral oil on the 
top of each reaction mixture. 

3. PCR products and plasmid DNA have to be cleaved by two different enzymes. Some 
enzymes are compatible (cut DNA with high efficiency using same buffer composition 
and conditions) and can be used simultaneously. If this is not the case or if one of the 
enzymes has low activity in the buffer used, we recommend that the DNA is cleaved 
separately with an additional purification step (use QIAquick PCR Purification Kit) 
between these two cleavages. 

4. Several methods are available for purification of both the PCR products and plasmids 
including electroelution, Freeze-Squeeze (Bio-Rad, Hercules, CA), GeneClean (Bio 101, 
La Jolla, CA), etc. Below, we describe the method we used in our lab, because this was 
fast, versatile (e.g., does not depend on DNA size) and DNA recovery is more than 95%. 
Both cleaved PCR products and plasmid are loaded onto 1% low-melting temperature 
agarose gel and run. If the volume of the sample is too large, use SpeedVac to decrease 
volume. Excise DNA fragment from the gel with a clean scalpel and transfer to a 1.5-mL 
microcentrifuge tube. Incubate tube for 10 min at 72°C (use waterbath) with occasional 
mixing. Transfer tube to preheated 42°C water bath quickly, add 3.5 uE AgaMC£ after 
2 min, and mix well. Incubate for an additional 45 min. Purify DNAs by QIAquick PCR 
Purification Kit following the manufacture's instruction. Use 50 uE of H 2 to elute 
DNAs. 

5. For routine cloning we use MAX Efficiency DH5a Competent Cells as they lack endonu- 
clease activity, accept large plasmid and increase insert stability owing to mutations in 
genes endAl, deoR, and recAl, respectively. 

Acknowledgment 

The author would like to thank Stephen Naylor for discussions and critical reading 
of the manuscript. 

References 

1. Chen, B. and Przybyla, E. A. (1994) An efficient site-directed mutagenesis method based 
on PCR. BioTechniques 17, 657-659. 

2. Sang, N., Condorelli, G., De Luca, A., MacLachlan, T. K., and Giordano, A. (1996) Gen- 
eration of site-directed mutagenesis by extra long, high-fidelity polymerase chain reac- 
tion. Analyt. Biochem. 233, 142-144. 

3. Tomic, M., Sunjevaric, I., Savtchenko, E. S., and Blumenberg, M. (1990) A rapid and 
simple method for introducing specific mutations into any position of DNA leaving all 
other position unalerted. Nucl. Acids Res. 18, 1656. 

4. Barik, S. (1993) Site-directed mutagenesis by double polymerase chain reaction: 
megaprimer method. Meth. Mol. Biol. 15, 277-286. 

5. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Site-directed 
mutagenesis by overlap extension using the polymerase chain reaction. Gene 11, 51-59. 

6. Zhong, D. and Bajaj, S. P. (1993) A PCR-based method for site specific domain replace- 
ment that does not require restriction recognition sequences. BioTechniques 15, 874-878. 

7. Kunkel, T. A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic 
selection. Proc. Nat. Acad. Sci. USA 82, 488-492. 



216 Adamec 

8. Striebel, H.-M., Rysavy, P., Adamec, J., Spizek, J., and Kalousek, F. (1996) Mutational 

analysis of both subunits from rat mitochondrial processing peptidase. Arch. Biochem. 

Biophys. 335,211-218. 
9 Adamec, J. and Kalousek, F. (1999) PCR method for generating multiple mutations at 

adjacent sites. Folia Microbiol. 44, 11-14. 
10. Moreira, R. F. and Noren, C. J. (1995) Minimum duplex requirements for restriction 

enzyme cleavage near the termini of linear DNA fragments. BioTechniques 19, 58-59. 



23 



A Fast Polymerase Chain Reaction-Mediated Strategy 
for Introducing Repeat Expansions 
into CAG-Repeat Containing Genes 

Franco Laccone 
1. Introduction 

Since their first description in 1991 (7), CAG-disease causing genes are increasing 
in number. Up to date, there are at least nine genetic diseases caused by CAG 
expansions. The creation of transgene and knock-in mice with CAG expansions is an 
useful tool for understanding the pathological mechanisms of the corresponding dis- 
eases, which could lead to therapeutic target(s) for the diseases. However, owing to 
the short life expectancy of the mice and to the low expression levels of transgenes, it 
is necessary to introduce CAG expansions larger than that in humans in order to elicit 
a pathological phenotype within the life of mice models. Naturally occurring "huge" 
CAG expansions (>100-150 CAGs), which could induce disease phenotype in the 
mice, are very seldom. In vitro synthesis of isolated CAG repeats have already been 
described (2,3). Most of these methods, however, require further cloning steps and 
often contain some flanking extraneous sequences. Here, the author describes a fast 
and simple way for expanding/introducing CAG repeats (or other repeats!) without 
altering the flanking 5' and 3' sequences of the gene of interest. This method was 
successfully employed for expanding the CAG repeat of the MJD/SCA3 gene (4). 
Fig. 1 outlined the strategy of this method. Two independent polymerase chain reac- 
tions (PCRs) amplify the target gene from 5' to the CAG repeat region (PCR I) and 
from the CAG repeat to the 3' region (PCR II) of the gene. The amplicons of PCR I and 
PCR II will be then mixed, elongated and then a third PCR will be carried out with the 
two most "outsider" primers. We used this strategy for elongating the CAG of the 
MJD/SCA3 gene from 22 up to 138 CAG repeats (see Figs. 2 and 3). This method can 
be used for elongating different repeats in different genes or even to insert and elon- 
gate any simple or complex repeat into a DNA sequence. However, it is not possible in 
this chapter to give specific conditions for each applications. It is important to adapt 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

217 



218 Laccone 

.-,-r, .. X CAG repeat y 
. target FOR-1 A K ' 

sequence — . , " ^ — ~~~ ,| 

REV-1 

B PCR I PCR II 

FOR-1 Y x < CAG >' 

=^m=>| H ■£ i -^L 

(<^G) 7 RE ^ 1 



1 - -*= — ' — » he 



FOR-1 
2. _^ 



o b d a □ a □ 



FOR-1 ( CTG ) 

3. -*- 



FOR-1 ( CTG ) 7 

4 T" . — ■■ ■■ _ 

(CTG) ; 



PCR I product PCR II product 



annealing and 
elongation step 



PCR III FOR-1 ^^ REV-1 

^ f ^M- I 



Fig. 1 . Strategy for introducing CAG expansions into CAG-containing genes. (A) Diagram- 
matic representation of the target sequence including the repeat region (CAG repeat), restric- 
tion sites X and Y, where X is the enzyme for the "5'-digestion" and Y is the enzyme for the 
"3 '-digestion" as explained in the text and the two outsider specific forward primer (FOR-1) 
and reverse primer (REV-1). (B) amplification of the target DNA in two distinct reactions after 
digestion with the X and Y restriction endonucleases in case of a circular template. As primers 
the pair FOR-l/(CTG)7 for PCR I and the pair (CTG)7/REV-1 for PCR II were used. (B2, B3, 
B4), subsequent cycles of PCR I leading to a progressive elongation of the repeat. (C) 
Amplicons of both PCRs. (D) Aliquots of both PCRs were mixed, denatured, annealed and 
extended with DNA polymerase. (E) Amplification of the elongated products of step D in PCR 
III with the specific (vector or gene specific) primers FOR-1 and REV-1. Arrowhead: variable 
CAG repeats. (Slightly modified from Laccone et al. (4), reprinted by permission of Wiley- 
Liss, Inc. Jossey-Bass Inc., a subsidiary of Wiley and Sons, Inc.) 



CAG-Repeat Expansions by PCR 

A B 



219 



12 3 4 



12 3 4 5 



AA 



CA 




AA- 

CA- 
PCRh 

PCR lie- 



Fig. 2. (A) Electrophoresis of the PCR products (7 uL each) on a 1% agarose gel of the 
MJD/SCA3 cDNA. Lane-1: 1-kb-ladder; Lane 2: amplification product of the complete 1.4-kb 
cDNA (CA, control amplification) obtained with the primers (FOR-1 and REV-1); Lane-3: 
PCR I product of about 1 . 1 kb (primers FOR- 1/(CAG)7); Lane 4: PCR II product obtained with 
primers primers (FOR-1 and REV-1); Lane-3: PCR I product of about 1.1 kb (primers FOR-1/ 
(CAG)7); Lane 4: PCR II product obtained with primers (CTG)7/REV-1. The visible smear in 
both amplicons of PCR I and II should represent most probably a continuous expansion of the 
repeat region. (B) Lane 1 : 1-kb ladder; lane 2: amplification of the target cDNA with the FOR-1 
and REV-1 specific primers. Lane 3-5: results of PCR III obtained by mixing 1, 2, and 4 uL of 
PCR I and PCR II amplicons with the two specific primers FOR-1 and REV-1. The visible 
smear represents most probably a series of products with elongated CAGs. In lane 2 the faint 
band of about 4.4 kb (AA = additional amplicons) may be caused by the linear amplification of 
the target circular clones which in this case have been not digested prior to the PCR. (From 
Laccone et al. (4), reprinted by permission of Wiley-Liss Inc. Jossey-Bass Inc., a subsidiary of 
Wiley and Sons, Inc.) 



this method to your own application, optimizing in particular the PCR conditions to 
your target. The required chemicals and instruments are usually available in every 
molecular genetic laboratory. 

2. Materials 

1. Gene of interest. 

2. Restriction endonucleases (In our case: Notl, Sail, EcoRV, New England Biolabs, Inc. 
Beverly, MA). 

3. Gene or vector specific primers. 

4. Repeat primers: in our case (CAG) 7 and (CTG) 7 . 

5. HotStarTaq Master Mix Kit (Qiagen, Bielefeld, Germany). 

6. pBluescript vector (Stratagene, La Jolla, CA). 

7. T4 DNA Ligase (Roche, Switzerland). 

8. Pfu polymerase (Stratagene). 

9. pSure Escherichia Coli competent cells (Stratagene). 

10. Plasmid isolation Mini Kit (Qiagen). 

11. Sequencing facilities. 

12. Basic devices of cloning experiments: electrophoresis chambers, centrifuges, PCR thermo 
cyclers, shakers and so on. 



220 



Laccone 




B 810111213 



* 1* 15 15 iTt&lfl ^2122 232* 25- * M 1616l7t*1fl MZ1ZZ2ar425 




:-J* 



*■ 2L2f LiB2¥3g31 37 333*35 36 * 26 27 2fi 263031 3?33 3435 39 





Fig. 3. (A) EcoRl/HindlH digestion of the cloned products of PCR III to release the plasmid 
insert. Because the MJD/SCA3 gene contains an EcoRI restriction site at position 435, we 
expected a constant fragment of about 450 bp (arrowhead) and a fragment of 700 + (CAG)n bp 
(arrow) attributable to the presence of CAGs variable in size. (B) Hybridzation of the blotted 
plasmid DNA with a probe containing 63 CAGs. (from Laccone et al. (4), reprinted by permis- 
sion of Wiley-Liss Inc. Jossey-Bass Inc., a subsidiary of Wiley and Sons, Inc.). 



3. Methods 

1. Digestion of circular target DNA (see Note 1). 

Two independent reactions containing each 1 (xg of plasmid and a restriction endonu- 
clease cutting at 3' and 5' end of the repeat region respectively in a volume of 50 u.L for 
linearizing the plasmid will be carried out. The digested products will be called for our 
convenience 3'-digestion and 5'-digestion, respectively. If the target DNA is linear, this 
step can be skipped. 20 [xL of each digested product are then diluted with 180 u,L H 2 in 
clean tubes to a concentration of about 1 ng/u,L. The rest of the digestion products should 
be stored at -20°C for further use. In case of linear DNA, the concentration of the DNA 
should be also of about 1 ng/(xL (see Fig. 1). 

2. PCR I and PCR II. 

In this step, the gene of interest will be amplified in two amplicons overlapping on the 
repeat region. 



CAG-Repeat Expansions by PCR 22 1 

a. PCR for 5' end (PCR I): 

1 jxL 3'-digestion product from step 1 

1 [xL specific forward primer (10 pmol/[xL) 

1 [xL (CTG) 7 as reverse primer (10 pmol/jxL) 

25 fxL HotstarTaq mix (Qiagen) 

22 [xL H 2 

b. PCR for 3' end (PCR II): 

1 [xL 5'-digestion product from step 1 

1 [xL specific reverse primer (10 pmol/|xL) 

1 u,L (CAG) 7 as forward primer (10 pmol/u,L) 

25 [xL HotstarTaq mix (Qiagen) 

22 [xL H 2 

c. Polymerase activation step: 

97°C for 15 min (polymerase activation step) 

d. PCR conditions for 30 cycles (see Note 2): 
96°C for 30 s 

55°C for 30 s 
72°C for 2 min 

3. Gel-electrophoresis of the amplicons of PCR I and PCR II. 

A successful amplification should show primary products of the expected size with a 
smear of larger products. After that, the products must be purified either with cold etha- 
nol precipitation or using commercial spin columns for removing unincorporated primers 
(see Fig. 2). 

4. Heteroduplex formation and elongation. 

Three reactions with different amount of amplicons from PCR I and II will be carried out: 

a. 1 (xL of amplicons from PCR I 

1 u,L of amplicons from PCR II 
25 fxL HotstarTaqmix (Qiagen) 
21 [xL H 2 

b. 2 |xL of amplicons from PCR I 

2 (xL of amplicons from PCR II 
25 [xL HotstarTaqmix (Qiagen) 
19 [xL H 2 

c. 4 |xL of amplicons from PCR I 
4 [xL of amplicons from PCR II 
25 |xL HotstarTaqmix (Qiagen) 
15 [xLH 2 

d. reaction conditions: 
97°C for 15 min 
60°C for 1 min 
72°C for 20 min 

5. PCR III. 

1 (xL of the "outsiders" Forward and Reverse primer (10 pmol/[xL) will be added to 
each reactions of step 4 above and the PCR will be carried out for 30 cycles at the follow- 
ing cycling conditions (see Notes 2 and 3): 96 C C for 30 s, 55°C for 30 s, 72°C for 2 min. 

The products of PCR III will be analyzed by agarose gel electrophoresis and the bands 
of interest will be cloned. 



222 Laccone 

6. Cloning strategies. 

In case the target gene was cloned into a plasmid and the outsiders primers were plasmid 
specific primers, it could be possible to cut the products with restriction endonucleases 
specific to the multiple cloning sites on the plasmid and subclone the digested product 
into a suitable vector. A more general method is to use a commercial T-vector according 
to the manufacturer's protocols or polishing the product with a proof-reading polymerase 
and cloning into blunt-ended vectors. This latter method will be described here because it 
is very convenient and economical. 

a. Digest 1 u,g of pBluescript II with EcoKV restriction endonuclease in a total volume 
of 50 [xL. After digestion 5 [xL will be diluted to a final concentration of 1 ng/uE by 
adding 95 [xL H 2 0. 

b. 20 u,L of amplicons of PCR III will be incubated with 0.5 u,L of Pfu polymerase 
(Stratagene) at 72°C for 30 min. 

c. Ligation: 

1 (xL pBluescript/EcoR V digestion(l ng/fxL) 

6 (xL polished amplicons (from step b) 

1 |xL T4 DNA ligase (Roche, Germany) 

1 uL 1 OX buffer 

1 [xL EcoR V restriction endonuclease (NEB) 

The ligation reaction will be carried out overnight at 14°C (see Note 4). 

d. "Killing" of religated vectors: 

Add 0.5 (xL of EcoR V restriction enzyme to the ligation reaction and incubate at 
37°C for 60 min. 

7. Transformation of the ligation reaction into suitable E. coli cells {see Note 5). 

8. Picking and analysis of colonies for the presence of inserts. Single white colonies should 
be put into liquid culture with the corresponding antibiotic (in our case ampicillin). Iso- 
late the plasmid using the Plasmid mini kit of Qiagen. Digestion of the positive plasmid 
and agarose gel electrophoresis analysis will reveal the size of the expansions (see Fig. 3). 

9. Sequence analysis of the recombinant clones. 

4. Notes 

1 . The digestion of the circular plasmid is very important to avoid a linear amplification of 
the original circular sequence. It is advisable to cut out the complete sequence of the gene 
of interest from the vector. 

2. The PCR conditions should most probably be adapted depending on the different target 
genes. The annealing temperature and elongation time are the two important variables 
that should be changed if required. As a general rule, it would be advisable to identify the 
optimal cycling conditions for the amplification of the complete gene with the two "out- 
siders" primers. Furthermore it would be advisable to develop cloning strategies for the 
amplification of products that are not very large (up to 2-kb each). The restriction map of 
the gene of interest should be helpful in identifying the desired positive clones (see Fig. 3A). 

3. It would be useful to amplify prior to PCR III the complete target with the "outsiders" 
primers for finding the optimal PCR conditions. In the PCR III the complete target should 
be amplified as a control of the PCR efficiency. 

4. The addition of EcoKV restriction endonuclease is advisable for reducing the background 
due to the vector's self-religation, provided that no EcoKV recognition sequence is con- 
tained within the gene of interest. An alternative to the EcoRV might be the Smal restric- 



CAG-Repeat Expansions by PCR 223 

tion endonuclease. However, the ligation into the Smal site was in our hands not so effi- 
cient as the EcoRV site. The ligation reaction will be carried out overnight at 14°C. 
5. Dealing with expanded CAG repeats requires caution. The most important one is to inhibit 
the transcription of the gene. As a matter of fact the transcription of elongated repeat can 
result in deletions or expansion of the repeat and furthermore can have a toxic effect on 
the cells. The amount of extractable plasmids from induced cells in this latter case 
decreases consistently. In our hand the pSure containing a F episome (F' proAB 
lac/ q ZAM15 TnlO [Tet 1 ]) has been very reliable. When preparing the competent cells in 
one's own laboratory it is very important to grow the pSure cells on plates containing 
tetracyclin. By omitting tetracyclin it might be possible that the cells will loss the F epi- 
some and the repression of the transcription by the lac/ q will be no longer efficient reduc- 
ing the amount of extractable plasmids per liquid cultures. 

The choice of the DNA polymerase for the amplification steps (PCR I, II and III) 
depends on the desired amplification fidelity. We used the Hotstar Taq DNA poly- 
merase as a compromise between fidelity and amplification efficiency. Using this poly- 
merase we have obtained clones up to 138 CAGs repeats without any errors and some 
clones with errors within the repeat. This random insertion of errors within the repeat 
(CAG to CCG or CAG to TAG) might be useful in some instances (e.g., effect of 
perfect versus imperfect repeats on cell culture or in mouse models). The use of a proof- 
reading polymerase, however, should reduce the error rate during the amplification. 

References 

1. La Spada, A. R., Wilson, E. M., Lubahn, D. B., Harding, A. E., and Fischbeck, K. H. 
(1991) Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. 
Nature 4, 77-79. 

2. Ordway, J. M. and Detloff, P. J. (1996) In vitro synthesis and cloning of long CAG repeats. 
Biotechniques 21, 609-612. 

3. Merry, D. E., Kobayashi, Y., Bailey, C. K., Taye, A. A., and Fischbeck, K. H. (1998) 
Cleavage, aggregation and toxicity of the expanded androgen receptor in spinal and bulbar 
muscular atrophy. Hum. Mol. Genet. 7, 693-701. 

4. Laccone, F., Maiwald, R., and Bingemann, S. (1999) A fast polymerase chain reaction- 
mediated strategy for introducing repeat expansions into CAG-repeat containing genes. 
Hum Mutat. 13, 497-502. 



24 



PCR Screening in Signature-Tagged Mutagenesis 
of Essential Genes 

Dario E. Lehoux and Roger C. Levesque 
1. Introduction 

Signature tagged-mutagenesis (STM) is a functional genomics technique that iden- 
tifies microbial genes required for infection within an animal host, or within host cell 
(1,2). As first described by Hensel et al., 1995 (3), transposon mutants are generated 
and each one tagged with a unique DNA sequence. Originally, STM used comparative 
hybridization to isolate mutants unable to survive in specified environmental condi- 
tions and to identify genes critical for survival in the host (3). The original STM has 
been modified to use defined oligonucleotides for tag construction into mini-Tn5 and 
to use polymerase chain reaction (PCR) instead of hybridization for rapid screening of 
bacterial mutants in vivo (4). The modified STM technique has been called PCR-based 
signature-tagged mutagenesis (PBSTM). 

STM is divided into two steps: the construction of a library of tagged mutants and 
the in vivo screening of the library. First, PBSTM scheme involves designing pairs (12 
in this case, but 24, 48, and 96 could be utilized) of 21-mers (see Table 1) synthesized 
as complementary DNA strands for cloning into the mini-Tn5 plasmid vector. The 
tagged minitransposons are used to mutagenize a microorganism. Each individual 
mutant can in theory be distinguished from every other mutant based on the different 
tag carried by the transposon in its genome (5). The set of 12 tags is repeatedly used to 
construct 12 libraries (see Fig. 1) and used for specific DNA amplifications easily 
detectable as signature tags (see Fig. 2). 

A key step in PCR is the design of primers with specific DNA sequence. In this 
goal, primers should be between 18-mers to 24-mers in length (6). Moreover, higher 
free energy for duplex formation (AG) (7) caused by insertion of certain nucleotides at 
the 5 '-end of a PCR primer stabilized primer-template duplex and optimized amplifi- 
cation reactions (8). On the other hand, the 3'-terminal position in primers was found 
essential for controlling mispriming (9). The insertion of a nucleotide mismatch at the 
3'- terminus of a primer-template duplex is more detrimental to PCR amplification 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

225 



226 Lehoux and Levesque 

Table 1 

DNA sequences of oligonucleotides synthesized for STM 

Tag number Nucleotide sequence" 

1 5'-GTACCGCGCTTAAACGTTCAG-3' 

2 GTACCGCGCTTAAATAGCCTG 

3 GTACCGCGCTTAAAAGTCTCG 

4 GTACCGCGCTTAATAACGTGG 

5 GTACCGCGCTTAAACTGGTAG 

6 GTACCGCGCTTAAGCATGTTG 

7 GTACCGCGCTTAATGTAACCG 

8 GTACCGCGCTTAAAATCTCGG 

9 GTACCGCGCTTAATAGGCAAG 

10 GTACCGCGCTTAACAATCGTG 

1 1 GTACCGCGCTTAATCAAG ACG 

12 GTACCGCGCTTAACTAGTAGG 

"Each 21-mers has a T m of 64°C and permits PCRs in one step when 
primer combinations are used for screening. The consensus 5'-ends 
comprising the first 1 3 nucleotides has higher AGs for optimizing PCRs. 
The variable 3'-ends indicated in bold define tag specificity and allow 
amplification of specific DNA fragments. Each tag is used as a primer in 
PCR with a primer synthesized within the Km resistant gene of mini-Tn5 
Km 2. The set of 12 21-mers representing the complementary DNA 
strand in each tag are not represented and can be deduced from the 
sequences presented. 



than internal mismatches (9), Specific oligonucleotides should be designed to optimize 
PCR and to have high specificity during screening by PCR. Twelve pairs of 21-mers 
were designed as tags following three basic rules: i) similar r m of 64°C to simplify tag 
comparisons by using one step of PCRs; ii) invariable 5'-ends with higher AG than at 
the 3'-end to optimize PCR amplification reactions; iii) variable 3'-end for an opti- 
mized yield of specific amplification product from each tag. The 21-mers are double 
stranded, and are cloned into a minitransposon (mini-Tn5 Km2) which is used to 
mutagenize and tag bacteria. All STM tags showed specificity as a unique DNA 
amplification product by PCR when using primers 1 to 12 in combination with the Km 
primer (see Fig. 2). 

A series of suicide plasmids carrying mini-Tn5s each with a specific tag are used to 
mutagenize targeted bacteria giving 12 libraries of mutants; 96 groups of 12 mutants 
are pooled and arrayed into 96-well plates (see Fig. 1). The 12 mutants from the same 
pool are grown separately overnight at 37°C. Aliquots of these cultures are pooled and 
a sample is removed for PCR analysis (the in vitro pool). A second sample from the 
same pool is used for the in vivo passage. After this passage, bacteria recovered from 
the animal organ (the in vivo pool) and the in vitro pool are used as templates in 12 
distinct PCR. Amplicons obtained with the in vitro and in vivo pools are compared. 



PCR Screening of STM Library 

Mumn khrur «hfc Eat I 



VlH-InJ»lll I ml 



227 



Mim-TK.^ " Irh I a\il 



M— Enf-nilh T«ill 




- :■ I. 



'.* rn- :>^: ::-r;-r i: 
at: jc jucx: 



n— n— ' nc: nc 



» rw* #F1I qiuiHi 
■kufcipM-irirticlDr 
Eh ™^^ arnyid mi I ] I *H- 



lilanli Ntrar- -nlrh li 



.' «nnon 

dHHCDl»I»D 



EKJLI uu-- 



Fig. 1. Construction of 12 libraries of P. aeruginosa mutants tagged with mini-Tn5 Km2. 
Double-stranded DNA tags were cloned into the pUTmini-Tn5 Knil plasmid (see methods). 
A r »!-resistant exconjugants were arrayed as libraries of 96 clones. In a defined library, each 
mutant has the same tag but inserted at different locations in the bacterial chromosome. One 
mutant from each library is picked to form 96 pools of 12 mutants with a unique tag for each. 
The differences between tags are represented by colors. O and I represent the 19 bps inverted 
repeats at each extremity of the mini-Tn5. 



Undetected mutants after the in vivo passage are in vivo attenuated. This simple STM 
method can be adapted to any bacterial system and used for genome scanning in 
various growth conditions. 

2. Materials 



9. 



10X medium salt buffer: 10 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 50 mM NaCl, 1 mM 

dithiothreitol (DTT). 

pUT mini-Tn5 Km2 plasmid (10). 

Kpn\ (New England Biolabs, Mississauga, Ont., Canada). 

10X NEB #1 buffer: 10 mM bis-Tris Propane-HCl, 10 mM MgCl 2 , 1 mM DTT, pH 7.0. 

10X bovine serum albumin (BSA) (1 mg/mL) (NEB). 

T 4 DNA polymerase (Gibco-BRL, Burlington, Ont., Canada) 

dNTPs (dATP, dGTP, dCTP, dTTP from Amersham Pharmacia Biotech, Baie d'Urfe, 

QC, Canada). 

T 4 DNA ligase 10X buffer (NEB): 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 

1 mM ATP, 25 Lig/mL BSA. 

T 4 DNA ligase (NEB). 



B 3 

do 



1 2 mulants wilh specific tag 

OOOIDOOOOOOD 







vs 




lllllllll 



Fig. 2. STM scheme for comparisons between the in vitro and in vivo negative selection step. Mutants from the same pool were grown 
as separate cultures. An aliquot was kept as the in vitro pool, and a second aliquot was used for injection into an animal model for in vivo 
selection. After this passage, bacteria were recovered from animal organs and constitute the in vivo pool. The in vitro and in vivo pools of 
bacteria were used to prepare DNA templates in 12 PCRs using the 21-mers 1 to 12 in Table 1 and the Km primer. PCR products were 
analyzed by agarose gel electrophoresis. Lanes 1 to 12: PCR products obtained with the primers 1 to 12. In this example, the mutant with 
Tagl 1 was not recovered after the in vivo selection. 



PCR Screening of STM Library 229 

10. Micropure-EZ pure (Millipore, Montreal, QC, Canada). 

11. Microcon 30 (Millipore). 

12. Microcon PCR (Millipore). 

13. Electrocompetent Escherichia coli Sll-lXpir (washed in glycerol 10%). 

14. 2-mm electroporation gap cuvets (BTX, distributed by VWR Can Lab, Mississauga, Ont., 
Canada), SOC (Fisher, Montreal, QC, Canada), 

15. SOC medium (Fisher): Formula per liter: 20 g Bacto tryptone, 5 g Bacto yeast extract, 
0.5 g sodium chloride, 2.4 g magnesium sulfate anhydrous, 0.186 g potassium chloride, 
20 mL of filter sterilized 20% glucose. 

16. Tryptic soy broth (TSB: Fisher): Formula per liter: 17 g Bacto tryptone, 3 g Bacto soytone, 
2.5 g Bacto dextrose, 5 g sodium chloride, 2.5 g dipotassium phosphate. 

17. Ampicillin (Sigma Chemical Company, St. Louis, MO). 

18. Kanamycin (Sigma). 

19. TE buffer: 10 mM Tris-HCl, pH 7.4, 0.1 mM ethylenediaminetetraacetic acid (EDTA) 
0.1 mM. 

20. PCR premix: 10X Taq polymerase (Gibco-BRL) reaction buffer without Mg 2+ : 200 mM 
Tris HC1, pH 8.4, 500 mM KC1, 50 mM MgCl 2 , 1.25 mM dNTPs, 10 pmoles oligonucle- 
otide tag (see Table 1), 10 pmoles pUTKanaRl (5-GCGGCCTCGAGCAAGACGTTT- 
'3), Taq polymerase (Gibco-BRL). 

21. Mineral oil. 

22. Agarose. 

23. IX Tris-borate EDTA buffer: 5X concentrated stock solution per liter: 54 g Tris base, 
27.5 g boric acid, 20 mL EDTA, pH 8.0. 

24. 0.5 (ig/mL ethidium bromide solution. 

25. Nylon membrane. 

26. Brain heart infusion agar (BHIA: Fisher). 

27. BHI (without agar). 

28. Sterile IX phosphate-buffered saline (PBS): 137 mMNaCl, 3 mMKCl, 10mMNa 2 HPO 4 , 
1.3 mM KH 2 P0 4 , pH 7.4. 

29. 2-mL 96-wells plates (Qiagen). 

30. 96-wells microtiter plates. 

31. Animals. 

32. Dissection kit. 

33. Potter homogenizer. 

34. QIAGEN genomic Tips (QUIAGEN). 

35. Selected endonuclease. 

36. pTZ18R (Amersham Pharmacia Biotech). 

37. Electrocompetent E. coli DH5a (washed in 10% glycerol). 

38. X-gal (Sigma). 

39. IPTG (Roche Diagnostics, Laval, QC, Canada). 

40. QIAGEN midi preparation kit. 

41. DNA sequencing service (Nucleic acids analysis and synthesis units, Laval University, 
http://www.rsvs.ulaval.ca). 

42. PC computer. 



230 Lehoux and Levesque 

3. Methods 

3.1. Construction of Tagged Mini-Tn5 Km2 

3.1.1. Double-stranded DNA tags 

1 . Twelve defined 2 1 -mers oligonucleotides should be synthesized along with their comple- 
mentary DNA strands as tags (see Table 1) (see Note 1) (Nucleic acids analysis and 
synthesis units, Laval University, http://www.rsvs.ulaval.ca.) 

2. 50 pmoles of both complementary oligonucleotides are mixed in 100 jxL of medium salt 
buffer for the annealing reactions. 

3. The annealing reaction mix is heated 5 min at 95°C, left to cool slowly at room tempera- 
ture in the block heater, and kept on ice (see Note 2). 

3.1.2. Minitransposon Tagging 

1. 20 [xg of pUT mini-Tn5 Km2 plasmid are digested with 20 units of Kpnl in 40 [xL of IX 
NEB #1 buffer with IX BSA (see Note 3). 

2. Incubate 2 h at 37°C. 

3. Inactivate 20 min at 65°C. 

4. 4 nmoles of each dNTPs and 5 units of T 4 DNA polymerase are added to digested plasmid 
solution to blunt extremities. 

5. Purify modified plasmid from enzymes with micropure-EZ and microcon 30 systems in a 
single step as described by the manufacturer's protocol. 

6. 0.04 pmoles of plasmid are ligated to 1 pmole of double stranded DNA tags in a final 
volume of 10 [xL of T 4 DNA ligase IX buffer containing 400 U of T 4 DNA ligase (see 
Note 4). 

7. Ligated products are purified using microcon PCR as described by the manufacturer's 
instructions and resuspended in 5 (xL of H 2 0. 

8. All the 5 [xL containing ligated products are electroporated in E. coli S17-l^pir (11) (see 
Note 5) using a Bio-Rad apparatus at 2.5 KV, 200 Ohms, 25 [xF in a 2-mm electroporation 
gap cuvet. After electroporation, 0.8 mL of SOC is added to cells, which are transferred 
in culture tubes to be incubated 1 h at 37°C. 

9. Transformants are selected on TSB supplemented with 50 |xg/mL of ampicillin and 
50 [xg/mL of kanamycin by plating 100 (xL of transformed cells. 

10. Single colonies are selected, purified and screened by colony PCR (see Note 6) in 50-uL 
reaction volumes containing: 10 (xL of boiled bacterial colonies in 100 uL of TE buffer; 
5 (xL of 10X Taq polymerase (Gibco-BRL) reaction buffer; 1.5 mM MgCl 2 ; 200 jxM of 
each dNTPs; 10 pmoles of one of the oligonucleotide tag (4) used to construct the DNA 
tags as a 5' primer and lOpmoles of the pUTKanaRl (5'-GCGGCCTCGAGCA- 
AGACGTTT-'3) as the 3' primer in the kanamycin resistance gene; 2.5 U of Taq poly- 
merase (Gibco-BRL). Thermal cycling conditions were (touchdown PCR) (see Note 7): a 
hot start for 7 min at 95°C, 2 cycles at 95°C for 1 min, 70 to 60°C for 1 min, and at 72°C 
for 1 min, then followed by 10 cycles at 95°C for 1 min, 60°C for 1 min, 72°C for 1 min 
in a DNA Thermal Cycler (Perkin Elmer Cetus). 10 fxL of the amplified products were 
analyzed by electrophoresis in a 1% agarose gel, IX Tris-borate EDTA buffer and stained 
for 10 min in 0.5 u.g/mL ethidium bromide solution (12). The amplicons should be around 
500 basepairs (see Fig. 2). 



PCR Screening of STM Library 23 1 

3.2. Tagged Mutants Libraries Construction 

3.2.1. Mutagenesis 

1 . E. coli S17-lXp(> strain (containing tagged pUT mini-Tn5 plasmid) is used as a donor for 
conjugal transfer into the recipient strain. Separate conjugation must be done for each 
tagged minitransposon. The dononrecipient ratio should be established to obtain the maxi- 
mum exconjugants by doing preliminary experiments. Cells are mixed and spotted as a 
50-uL drop on a nylon membrane placed on a nonselective BHIA plate. Plates are incu- 
bated at 30°C for 8 h (see Note 8). 

2. Filters are washed with 10 mL of phosphate buffered saline (PBS) to recover bacteria. 

3. Five 100 u,L aliquots of the PBS solution containing exconjugants are plated on five 
BHIA plates supplemented with the appropriate antibiotic to select for the strain. Kana- 
mycin is used to select exconjugants with the mini-Tn5 inserted into their chromosomes 
(see Note 9). Plates are incubated overnight at 37°C. 

4. Exconjugants are selected on BHIA supplemented with ampicillin. Mutants resistant to 
ampicillin are removed from the pool, since they carry the suicide vector inserted into the 
chromosome. 

5. Kanamycin resistant and ampicillin sensitive exconjugants from a single conjugation are 
arrayed as a library of 96 clones in 2 mL 96-wells plate in 1 .5 mL of BHI supplemented 
with kanamycin and appropriate antibiotic. The 2-mL 96-wells plates are incubated 
18-22 h at 37°C (see Note 10). At this step, 12 differently tagged libraries are obtained. 

6. As an STM working scheme, one mutant from each library is picked to form 96 pools of 
12 unique tagged mutants (see Fig. 1) contained in the 2-mL 96-wells plates. 

3.3. In Vivo Screening of Tagged Mutants 

1. Each mutant from the same pool are inoculated individually in 200 uL of TSB containing 
kanamycin and grown overnight at 37°C without agitation in microtiter plate. 

2. Aliquots of these cultures are pooled. 

3. A first sample is diluted from 10 _1 to 10" 4 , and plated on BHIA supplemented with 
kanamycin. 

4. After overnight incubation at 37°C, 10 4 colonies are recovered in 5 mL of PBS and a 
sample of 1 mL is removed for PCR and called the in vitro pool. 

5. The 1 mL in vitro pool sample is spun down and the cell pellet is resuspended in 1 mL of 
TE buffer. 

6. The in vitro pool is boiled 10 min, spun down, and 10 (xL of supernatant are used in PCR 
analysis as described above. 

7. A second sample from the pooled cultures is used to inoculate animals. 

8. After the appropriate in vivo incubation time, animals are sacrificed and bacteria are 
recovered from the targeted organs (see Note 11). 

9. Tissues are recovered by dissection and homogenized with a Potter homogenizer in 
10 mL of sterile phosphate buffered saline pH 7.0 contained in a 50-mL falcon tube (see 
Note 12). 

10. 100 L of homogenized tissues are plated on BHIA supplemented with kanamycin. After 
the in vivo selection, 10 4 colonies recovered from a single plate are pooled in 5 mL of 
PBS. From the 5 mL, 1 mL is spun down and resuspended in 1 mL of TE buffer (the in 
vivo pool). 



232 Lehoux and Levesque 

11. The in vivo pool is boiled 10 min, spun down, and 10 uL of supernatant is used in PCR 
analysis as described above. 10 uL of PCR are used for 1% agarose gel electrophoresis 
separation. 

12. PCR amplification products of tags present in the in vivo pool are compared with ampli- 
fied products of tags present in the in vitro pool (see Fig. 2). 

13. Mutants that give PCR amplicon from in vitro pool and not from in vivo pool are purified 
and kept for further analysis (see Note 13). 

3.4. Cloning and Sequence Analysis of Transposon-Flanking DNA 
from Attenuated Mutants 

1. Chromosomal DNA from attenuated mutants is prepared using the Qiagen genomic DNA 
extraction kit as described in the manufacturer's protocol. 

2. Chromosomal DNA (1 u,g) is digested with endonuclease giving a large range of frag- 
ment sizes (see Note 14). 

3. Digested chromosomal DNAs are cloned into pTZ18R predigested with the correspond- 
ing endonuclease. Ligation reactions are done as follows: 1 [xg of digested chromosomal 
DNA is mixed with 50 ng of digested pTZ18r in 20 uL of IX T 4 DNA ligase buffer with 
40 units of T 4 DNA ligase. 

4. Incubate overnight at 16°C. 

5. Ligated products are purified using microcon PCR as described by the manufacturer's 
instructions and resuspended in 5 [xL of H 2 0. 

6. The 5-uL recombinant plasmid solution is used for electroporation in E. coli DH5a as 
described previously. 

7. All the electroporation cells are spun down and resuspended in 100 uL of BHI to be 
plated on a selective plate. Recombinant bacteria are selected as white versus blue colo- 
nies on X-gal/IPTG containing plates (0.005% and 0.1 mM, respectively) with ampicillin 
(100 [xg/mL) and kanamycin (50 [Xg/mL) (see Note 15). 

8. Clones are kept and purified for plasmid analysis. 

9. Plasmid DNAs are prepared with QIAGEN midi preparation kit as described by the manu- 
facturer. 

10. These plasmids are sequenced using the complementary primer of the corresponding 
tagged mutant. Automated sequencing (ABI 373) is done as suggested by the manu- 
facturer. 

1 1 . DNA sequences obtained are assembled and subjected to database searches using BLAST 
included in the GCG Wisconsin package (version 10.0). Complete open reading frames 
(ORF) of disrupted genes and similarity searches with complete genomes can be per- 
formed at NCBI using the microbial genome sequences (http://www.ncbi.nlm.nih.gov). 

4. Notes 

1. Here we present an example of a set of twelve 21-mers as DNA tags. However, it is 
possible to elaborate more or other DNA tags by following previously described rules. 
Specificity and quality of amplification should be tested prior to using them for tagged 
minitransposon in a mutagenesis experiment because it should has no cross amplification 
from different DNA tags. 

2. The annealing oligonucleotide mixture should be made before each ligation. 

3. It is possible to digest several small quantities of DNA preparation and after purification 
(step 3.1.2.5.) pool all digested plasmid preparations. 



PCR Screening of STM Library 233 

4. Using freshly made annealing oligonucleotide mixture raises the efficiency of ligation 
because tags might be degraded. 

5. For replication and maintenance of the recombinant plasmid, it might be useful to use the 
well-known E. coli DH5akpir strain. However, it will be necessary to transfer plasmids 
in the Sll-lXpir strain to transfer DNA by conjugation. 

6. It might be necessary to screen several colonies to find the good recombinant. It is pos- 
sible to pool several colonies to reduce the number of PCRs (13). This ensures that you 
have the good recombinant among the selected colonies in very few PCRs. To bypass the 
necessity of doing plasmid preparations, PCRs can be done on bacterial cell lysates. One 
or several colonies are resuspended in 100 u.L of TE buffer, boiled 10 min, and spun 
down. 10 [xL of supernatant are used for the PCR template. After the pool PCR, the spe- 
cific clone containing tagged plasmid should be identified within the pool. 

7. Touchdown PCR was preferred to the standard PCR cycle because establishment of opti- 
mal PCR conditions for two primers is facilitated, and it increased specificity of amplifi- 
cation products obtained . It involves decreasing the annealing temperature by 1°C every 
second cycle to a "touchdown" annealing temperature, which is then used for 10 or so 
cycles. In this case, annealing temperature takes place at 6°C above the calculated T m . 
During the following cycles, the annealing temperature is gradually reduced by 1°C until 
it has reached a level of approx 4 C C below T m . 

8. Temperature and incubation time should be determined by preliminary experiments. 

9. It is very important to use the good kanamycin concentration to eliminate background 
related to the inoculum effect. Minimal inhibitory concentration can be determined to 
evaluate the effective kanamycin concentration. 

10. In a defined library, each mutant has the same tag but is assumed to be inserted at a 
different location in the bacterial chromosome. Southern blot hybridization is necessary 
to confirm the random integration of the mini-Tn5 (12). 

11. Parameters concerning animal model should be particularly well defined. The inoculum 
size necessary to cause infection determines the complexity of mutants pooled. In fact, 
each mutant in a defined input pool has to be in a sufficient cell number to initiate infec- 
tion. The inoculum size must not be too high, resulting in the growth of mutants which 
would otherwise have not been detected (2). Other important parameters in STM include 
the route of inoculation and the time-course of a particular infection. Also, certain gene 
products important directly or indirectly for initiation or maintenance of the infection 
may be niche-dependent or expressed specifically in certain animal or plant tissues only. 
If the duration of the infection in STM in vivo selection is short, genes important for 
establishment of the infection will be found, and if the duration is long, genes important 
for maintenance of infection will be identified (2). Several routes of inoculation and dif- 
ferent animal models can be used. 

12. Keep homogenates on ice. 

13. Each STM attenuated mutant has to be confirmed by: a second round of STM screening 
(14), comparisons between in vivo bacterial growth rate of mutants versus growth of the 
wild-type in single (15) or competitive (16) infections, or estimation of LD 50 (3). 

14. More than one endonuclease or partial digestion can be used to obtain more DNA frag- 
ments ranging from 1 to 4 Kb that are easier to clone in pTZ18r. 

15. Only clones that contain plasmid with chromosomal fragments and the mini-Tn5 marker 
are obtained. 



234 Lehoux and Levesque 

References 

1. Shea, J. E., Santangelo, J. D., and Feldman, R. G. (2000) Signature-tagged mutagenesis in 
the identification of virulence genes in pathogens. Curr. Opin. Microbiol. 3, 451-458. 

2. Lehoux, D. E. and Levesque, R. C. (2000) Detection of genes essential in specific niches 
by signature-tagged mutagenesis. Curr. Opin. Biotechnol. 11, 434-439. 

3. Hensel, M., Shea, J. E., Gleeson, C, Jones, M. D., Dalton, E., and Holden, D. W. (1995) 
Simultaneous identification of bacterial virulence genes by negative selection. Science 
269, 400-403. 

4. Lehoux, D. E., Sanschagrin, F., and Levesque, R. C. (1999) Defined oligonucleotide tag 
pools and PCR screening in signature-tagged mutagenesis of essential genes from bacte- 
ria. Biotechniques 26, 473-478, 480. 

5. Chiang, S. L., Mekalanos, J. J., and Holden, D. W. (1999) In vivo genetic analysis of 
bacterial virulence. Annu. Rev. Microbiol. 53, 129-154. 

6. Dieffenbach, W. C. and Dveksler, G. S., eds. (1995) PCR Primer: A Laboratory Manual. 
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 714. 

7. Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A. (1986) Predicting DNA duplex 
stability from the base sequence. Proc. Natl. Acad. Sci. USA 83, 3746-3750. 

8. Rychlik, W. (1993) Selection of primer chain reaction, in PCR protocols: current applica- 
tion, W.H. Press, Editor.: Totowa NJ. 

9. Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C, and Sninsky, 
J. J. (1990) Effects of primer-template mismatches on the polymerase chain reaction: 
human immunodeficiency virus type 1 model studies. Nucl. Acids Res. 18, 999-1005. 

10. De Lorenzo, V., Herrero, M., Jakubzik , U., and Timmis, K. N. (1990) Mini-Tn5 
transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal 
insertion of cloned DNA in Gram-negative eubacteria. J . Bacteriol. 172, 6568-6572. 

11. Simon, R., Priefer, U., and Piihler, A. (1983) A broad range mobilization system for in 
vitro genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Tech- 
nology 1,784-791. 

12. Sambrook, J., Fritsch, E. F., and Maniatis, T., (eds) (1989) Molecular Cloning: A Labora- 
tory Manual, 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

13. Dewar, K., Sabbagh, L., Cardinal, G., Veilleux, F., Sanschagrin, F., Birren, B., and 
Levesque, R. C. (1998) Pseudomonas aeruginosa PAOl bacterial artificial chromosomes: 
strategies for mapping, screening, and sequencing 100 kb loci of the 5.9 Mb genome. 
Microb. Comp. Genom. 3, 105—117. 

14. Darwin, A. J. and Miller, V. L. (1999) Identification of Yersinia enterocolitica genes af- 
fecting survival in an animal host using signature-tagged transposon mutagenesis. Mol. 
Microbiol. 32, 51-62. 

15. Camacho, L. R., Ensergueix, D., Perez, E., Gicquel, B., and Guilhot, C. (1999) Identifica- 
tion of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged 
transposon mutagenesis. Mol. Microbiol. 34, 257-267 '. 

16. Chiang, S. L. and Mekalanos, J. J. (1998) Use of signature-tagged transposon mutagenesis 
to identify Vibrio cholerae genes critical for colonization. Mol. Microbiol. 27, 797-805. 



25 

Staggered Extension Process (StEP) 
In Vitro Recombination 

Anna Marie Aguinaldo and Frances Arnold 
1. Introduction 

In vitro polymerase chain reaction ( PCR)-based recombination methods are used 
to shuffle segments from various homologous DNA sequences to produce highly 
mosaic chimeric sequences. Genetic variations created in the laboratory or existing in 
nature can be recombined to generate libraries of molecules containing novel combi- 
nations of sequence information from any or all of the parent template sequences. 
Evolutionary protein design approaches, in which libraries created by in vitro recom- 
bination methods are coupled with screening (or selection) strategies, have success- 
fully produced variant proteins with a wide array of modified properties including 
increased drug resistance (1,2), stability (3-6), binding affinity (6), improved folding 
and solubility (7), altered or expanded substrate specificity (8,9), and new catalytic 
activity (10). 

Stemmer reported the first in vitro recombination, or "DNA shuffling," method for 
laboratory evolution (//). An alternative method called the staggered extension pro- 
cess (StEP) (12) is simpler and less labor intensive than DNA shuffling and other 
PCR-based recombination techniques that require fragmentation, isolation, and ampli- 
fication steps (1,11,13,14). StEP recombination is based on cross hybridization of 
growing gene fragments during polymerase-catalyzed primer extension (12). Follow- 
ing denaturation, primers anneal and extend in a step whose brief duration and subop- 
timal extension temperature limit primer extension. The partially extended primers 
randomly reanneal to different parent sequences throughout the multiple cycles, thus 
creating novel recombinants. The procedure is illustrated in Fig. 1. The full-length 
recombinant products can be amplified in a second PCR, depending on the product 
yield of the StEP reaction. The StEP method has been used to recombine templates 
with sequence identity ranging from single base differences to natural homologous 
genes that are approx 80% identical. 



From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 



235 



236 



Aguinaldo and Arnold 



-#• 


1 




1 


□ 


1 1 



Priming 



Extension 



Denaturation followed by random priming and 
extension 



□ 



I Repeated cycles 

I 




Fig. 1. StEP recombination, illustrated for two gene templates. Only one primer and single 
strands from the two genes (open and solid blocks) are shown for simplicity. During priming, 
oligonucleotide primers anneal to the denatured templates. Short fragments are produced by 
brief polymerase-catalyzed primer extension that is interrupted by denaturation. During subse- 
quent random annealing-abbreviated extension cycles, fragments randomly prime the templates 
(template switching) and extend further, eventually producing full-length chimeric genes. The 
recombinant full-length gene products can be amplified in a standard PCR (optional). 



2. Materials 

1. DNA templates containing the target sequences to be recombined (see Note 1). 

2. Oligonucleotide primers universal to all templates to be recombined (see Note 2). 

3. Taq DNA polymerase (see Note 3). 

4. 10X PCR buffer: 500 mM KC1, 100 mM Tris-HCl, pH 8.3. 

5. 25mMMgCl 2 . 

6. dNTP solution: 10 mM of each dNTP. 

7. Agarose gel electrophoresis supplies and equipment. 

8. Dpnl restriction endonuclease (20 U/uE) and 10X supplied buffer (New England Biolabs, 
Beverly, MA). 

9. QIAquick gel extraction kit (Qiagen, Valencia, CA) or your favorite method. 



StEP In Vitro Recombination 237 

3. Methods 

1 . Combine 1-20 ng total template DNA, 0. 15 \»M each primer, IX PCR buffer, 200 yM dNTP 
mix, 1.5 mM MgCl 2 , 2.5 U Taq polymerase, and sterile dH 2 to 50 uL. Set up a negative 
control reaction containing the same components but without primers (see Note 4). 

2. Run the extension protocol for 80-100 cycles using the following parameters: 94°C for 
30 s (denaturation) and 55°C for 5-15 s (annealing/extension) (see Notes 5 and 6). 

3. Run a 5-10 jaL aliquot of the reactions on an agarose gel to check the quality of the 
reactions (see Note 7). If a discrete band with sufficient yield for subsequent cloning is 
observed after the StEP reaction, and the size of the full-length product is clearly distin- 
guishable and easily separated from the original starting templates, proceed to step 8. 

4. If parental templates were purified from a dam+ Escherichia coli (E. Coli) strain (see 
Note 1), combine 2 \ih of the StEP reaction, IX Dpnl reaction buffer, 5-10 U Dpnl 
restriction endonuclease, and sterile dH 2 to 10 uL. Incubate at 37°C for 1 h (see Note 8). 

5. Amplify the target recombinant sequences in a standard PCR using serial dilutions (1 uL 
of undiluted, 1:10, 1:20, and 1:50 dilutions) of the Dpnl reaction (or the StEP reaction if 
Dpnl digestion was not done). Mix IX PCR buffer, 1.5 mM MgCl 2 , 200 \iM dNTP mix, 
20 \lM of each primer, 2.5 U Taq DNA polymerase, and sterile dH 2 to 100 uL. 

6. Run the amplification reaction for 25 cycles using the following parameters: 94°C for 
30 s, 55°C for 30 s, and 72 C for 60 s for each 1 kb in length. 

7. Run a 10-uL aliquot of the amplified products on an agarose gel to determine the yield 
and quality of amplification (see Note 9). Select the reaction with high yield and low 
amount of nonspecific products. 

8. Gel purify the desired full-length reaction product following the manufacturer's protocol 
in the QIAquick gel purification kit. Digest the purified fragment with the appropriate 
restriction endonucleases for ligation into the preferred cloning vector. 

4. Notes 

1 . Appropriate templates include plasmids carrying target sequences, sequences excised by 
restriction endonucleases and PCR amplified sequences. Reactions are more reproduc- 
ible as template size decreases because this reduces the likelihood of nonspecific prim- 
ing. For example, three 8.5-kb plasmid templates containing different 1.7-kb target 
sequences were less efficient for StEP recombination than 3-kb restriction fragments con- 
taining the target sequences. Unusually large plasmids and templates should be avoided. 

Short template lengths may also pose a problem when the size is indistinguishable in 
length from the desired product. Conventional physical separation techniques, such as 
agarose gel electrophoresis, cannot be used to isolate the reaction product from the tem- 
plate, resulting in a high background of nonrecombinant clones. To minimize parental 
templates that may contribute to background nonrecombinant clones, plasmids used for 
template preparations (both intact plasmids and restriction fragments containing target 
sequences excised from plasmids) should be isolated from a methylation positive E. coli 
strain, e.g., DH5a (BRL Life Technologies, Gaithersburg, MD) or XLl-Blue (Stratagene, 
La Jolla, CA). These dam+ strains methylate DNA. Dpnl, a restriction endonuclease that 
cleaves methylated GATC sites, can then be used to digest parental templates without 
affecting the PCR products. 

2. Primer design should follow standard criteria including elimination of self-complementarity 
or complementarity of primers to each other, similar melting temperatures (within approx 
2-4°C is best), and 40-60% G + C content. Primers of 21-24 bases in length work well. 



238 Aguinaldo and Arnold 

3. Other investigators have used Vent DNA polymerase instead of Taq DNA polymerase in 
StEP recombination (IS). Vent DNA polymerase is one of several thermostable DNA 
polymerases with proofreading activity leading to higher fidelity (16). Use of these alter- 
native polymerases is recommended for DNA amplification when it is necessary to mini- 
mize point mutations. In addition, the proofreading activity of high-fidelity polymerases 
slows them down, offering an additional way to increase recombination frequency (17). 
Vent polymerase, for example, is reported to have an extension rate of 1000 nucleotides/ 
min and processivity of 7 nucleotides/initiation event as compared to the higher 4000 
nucleotides/min and 40 nucleotides/initiation event of Taq DNA polymerase (18). Slower 
rates lead to shorter extension fragments and greater crossover frequency. 

4. The negative control reaction should be processed in the same manner as the sample 
reactions for all the steps of the procedure. No product should be visible for the no primer 
control throughout the procedure. If bands are present in the negative control similar to 
the sample reactions, products of the sample reactions may be the result of template con- 
tamination resulting in nonrecombinant clones. 

5. The annealing/extension times chosen are based on the number of crossover events desired. 
Shorter extension times as well as lower annealing temperatures lead to increased numbers 
of crossovers due to the shorter extension fragments produced for each cycle. The size of 
the full-length product determines the number of reaction cycles. Longer genes require a 
greater number of reaction cycles to produce the full-length genes. The annealing tempera- 
ture should be a few degrees lower than the melting temperature of the primers. 

6. The progression of the fragment extensions can be monitored by taking 10 jiL aliquots 
of a duplicate StEP reaction at defined cycle numbers and separating the fragments on 
an agarose gel. For example, samples taken every 20 cycles from StEP recombination 
of two subtilisin genes showed reaction product smears with average sizes approaching 
100 bp after 20 cycles, 400 bp after 40 cycles, 800 bp after 60 cycles, and a clear dis- 
crete band around 1 kb (the desired length) within a smear after 80 cycles (12). DNA 
polymerases currently used in DNA amplification are very fast. Even very brief cycles 
of denaturation and annealing provide time for these enzymes to extend primers for 
hundreds of nucleotides. For Taq DNA polymerase, extension rates at various tempera- 
tures are: 70°C, > 60 nt/s; 55°C, approx 24 nt/s; 37°C, approx 1.5 nt/s; 22°C, approx 
0.25 nt/s (19). Therefore, it is not unusual for the full-length product to appear after 
only 10-15 cycles. The faster the full-length product appears in the extension reaction, 
the fewer the template switches that have occurred and the lower the crossover fre- 
quency. To increase the recombination frequency, everything possible should be done to 
minimize time spent in each cycle: selecting a faster thermocycler, using smaller test 
tubes with thinner walls, and, if necessary, reducing the reaction volume. 

7. Possible reaction products are full-length amplified sequence, a smear, or a combination 
of both. Appearance of the extension products may depend on the specific sequences 
recombined or the template used. Using whole plasmids may result in nonspecific anneal- 
ing of primers and their extension products throughout the vector sequence, which can 
appear as a smear on the gel. A similar effect may be observed for large templates. If no 
reaction products are visible, the annealing/extension times and the temperature of the 
StEP reaction will need to be determined empirically. Try reducing the annealing tem- 
peratures as well as modifying the primer and/or template concentrations. 

8. The background from non-recombinant clones can be reduced following the StEP reac- 
tion by Dpnl endonuclease digestion to remove methylated parental DNA (see Note 1). 
At this point you want to get rid of the DNA template that is still in your reaction mixture 
before proceeding to amplification to prevent carryover contamination. 



StEP In Vitro Recombination 239 

9. If the amplification reaction is not successful and you get a smear with a low yield of 
full-length sequence, reamplify these products using nested internal primers separated 
by 50-100 bp from the original primers. 

References 

1. Stemmer, W. P. (1994) DNA shuffling by random fragmentation and reassembly: in vitro 
recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 81, 10,747-10,751. 

2. Crameri, A., Raillard, S. A., Bermudez, E., and Stemmer, W. P. C. (1998) DNA shuffling of 
a family of genes from diverse species accelerates directed evolution. Nature 391, 288-291. 

3. Giver, L., Gershenson, A., Freskgard, P. O., and Arnold, F. A. (1998) Directed evolution 
of a thermostable esterase. Proc. Natl. Acad. Sci. USA 95, 12,809-12,813. 

4. Zhao, H. M. and Arnold, F. H. (1999) Directed evolution converts subtilisin E into a func- 
tional equivalent of fhermitase. Protein Eng. 12, 47-53. 

5. Miyazaki, K., Wintrode, P. L., Grayling, R. A., Rubingh, D. N., and Arnold, F. H. (2000) 
Directed evolution study of temperature adaptation in a psychrophilic enzyme. ./. Mol. 
Biol. 297, 1015-1026. 

6. Jermutus, L., Honegger, A., Schwesinger, F., Hanes, J., and Pluckthun, A. (2001) Tailoring 
in vitro evolution for protein affinity or stability. Proc. Natl. Acad. Sci. USA 98, 75-80. 

7. Waldo, G. S., Standish, B. M., Berendzen, J., and Terwilliger, T. C. (1999) Rapid protein- 
folding assay using green fluorescent protein. Nat. Biotech. 17, 691-695. 

8. Zhang, J. H., Dawes, G., and Stemmer, W. P. C. (1997) Directed evolution of a fucosidase 
from a galactosidase by DNA shuffling and screening. Proc. Natl. Acad. Sci. USA 94, 
4504-4509. 

9. Kumamaru, T., Suenaga, H., Mitsuoka, M., Watanabe, T., and Furukawa, K. (1998) 
Enhanced degradation of polychlorinated biphenyls by directed evolution. Nat. Biotech. 
16, 663-666. 

10. Altamirano, M. M., Blackburn, J. M., Aguayo, C, and Fersht, A. R. (2000) Directed evo- 
lution of new catalytic activity using the alpha/beta-barrel scaffold. Nature 403, 617-622. 

11. Stemmer. W. P. C. (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 
370, 389-391. 

12. Zhao, H., Giver, L., Shao, Z., Affholter, J. A., and Arnold, F. H. (1998) Molecular evolution 
by staggered extension process (StEP) in vitro recombination. Nat. Bioteclmol. 16, 258-261. 

13. Shao, Z., Zhao, H., Giver, L., and Arnold, F. H. (1997) Random-priming in vitro recombi- 
nation: an effective tool for directed evolution. Nucl. Acids Res. 26, 681-683. 

14. Volkov, A. A. and Arnold, F. H. (2000) Methods for in vitro DNA recombination and 
random chimeragenesis. Meth. Enzymol. 28, 447-456. 

15. Ninkovic, M., Dietrich, R., Aral, G., and Schwienhorst, A. (2001) High-fidelity in vitro 
recombination using a proofreading polymerase. BioTechniques 30, 530-536. 

16. Cline, J., Braman, J. C, and Hogrefe, H. H. (1996) PCR fidelity of Pfu DNA polymerase 
and other thermostable DNA polymerases. Nucl. Acids Res. 24, 3546-3551. 

17. Judo, M. S. B., Wedel, A. B., and Wilson, C. (1998) Stimulation and suppression of PCR- 
mediated recombination. Nucl, Acids Res. 26, 1819-1825. 

18. Kong, H., Kucera, R. B., and Jack, W. E. (1993) Characterization of a DNA polymerase 
from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady 
state kinetics, thermal stability, processivity, strand displacement, and exonuclease activi- 
ties. ./. Biol. Chem. 268, 1965-1975. 

19 Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. D. (1988) DNA sequencing 
with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain 
reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 9436-9440. 



26 

Random Mutagenesis 

by Whole-Plasmid PCR Amplification 

Donghak Kim and F. Peter Guengerich 

1. Introduction 

1.1. General Introduction 

Mutagenesis is a popular tool used in the analysis of protein structure and function. 
Polymerase chain reaction (PCR)-based mutagenesis can be used to introduce muta- 
tions with the use of the appropriate primer. Although the majority of attention has 
been given to site-directed mutagenesis, random mutagenesis is actually an older 
approach and has considerable potential because of its limited bias, if an appropriate 
screening method is available. This approach has successfully been used to obtain 
"gain-of- function" mutants (1). The ability to target mutants to individual proteins and 
parts of proteins with modern molecular tools has considerable applicability. 

Generation of reliable random libraries for screening presents a particular chal- 
lenge. Ideally, all potential clones should be represented in the library. Conventional 
methods include duplex oligonucleotide cassette synthesis followed by subcloning 
between two unique restriction sites (2), degenerate PCR-based methods using Mn 2+ 
or splicing by overlap extension (3,4), single-stranded mutagenesis using bacterioph- 
age M13-based vectors (5), and various chemical methods (6,7). Depending on the 
technique employed, introduction of multiple unique restriction sites for subcloning 
or single-stranded DNA rescue is often required. Other disadvantages of some of these 
methods include mutations outside the targeted region, intolerably high background 
from the native sequence, and mutational bias in terms of the types of nucleotide sub- 
stitutions observed (8). 

1.2. PCR-Based Whole Plasmid Amplification 

We describe a less cumbersome method for random mutagenesis of up to five 
consecutive amino acids within a protein by PCR-based whole plasmid amplification 
using complementary degenerate primers. This method was originally introduced from 



From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 



241 



242 



Kim and Guengerich 



Table 1 

Primer Codons for Random Mutagenesis 

with No Wild-Type Background Remaining 



D 



H 



I 



K 



amino A C 

acid 
mutation SHN YNN YNN RNN YNN SDN YNN SBN RNN SBN 
primer 



M 



N 



Q 



R 



V 



w 



amino 
acid 
mutation DNN YNN SHN RNN RDN SWN SHN SBN DNN YNN 
primer 

The mutation primers represent the antisense primers from 5' to 3'. The sense primer can be designed 
as complementary. IUB Codes for Bases (N = G, A, T, or C in equal amount; S = G or C in equal 
amount; Y = C or T in equal amount; R = A or G in equal amount). 



this laboratory (9), based on a modification of the Stratagene® QuikChange™ Site- 
Directed Mutagenesis approach. Also, a restriction enzyme-marker site is introduced 
to exclude the wild-type gene (10). The combination of reliability, convenience, low 
cost, and wide applicability of this technique renders it very practical for the construc- 
tion of mutant libraries randomized within a limited target zone. 

2. Materials 

1 . Oligonucleotides: Sequences of oligonucleotide primer pairs for random mutagenesis can 
be chosen according to the following criteria: 

a. Sense and antisense primers are of equal length, with the same start and end positions 
with respect to the coding strand of the template DNA (see Note 1). 

b. Codons in the target region for random mutagenesis are encoded as in Table 1 (see 
Note 2). 

c. Primers encompass the desired target region with a minimum of 12 bp of wild-type 
sequence on either side of the mismatches, preferentially terminating in multiple G or 
C residues to anchor the primer (see Note 3). 

d. In cases where a unique restriction site is present within the template plasmids, the 
oligonucleotide is extended beyond the restriction site by the number of bases required 
for efficient (>90%) cleavage of the linear PCR product (see Fig. 1). 

e. In cases where no unique site is present, a restriction site is incorporated into the 
primer by silent mutagenesis with the primer length increased correspondingly to 
ensure annealing (see Note 4). 

f. Oligonucleotides for mutagenesis are synthesized on a 200 nmol scale and then puri- 
fied with the appropriate methods. 

2. Plasmids: Small plasmids (containing the cDNA insert of interest) such as pBluescript® 
plasmid DNA (Stratagene, La Jolla, CA) are preferred for efficient PCR. 

3. DNA Polymerase and PCR Buffer: 2.5 U native pfu DNA polymerase (Stratagene); pfu 
DNA polymerase buffer: 20 mM Tris-HCl (pH 8.75), 10 mM KC1, 10 mM (NH 4 ) 2 S0 4 , 
2 mM MgS0 4 , 0.10% Triton X-100 (w/v), and 100 ug BSA/mL (see Note 5). 



Random Mutagenesis 



243 



X Ba/nHI 

5 'GGAAAACACCAAGNNNNNNNNNNNNTTTGATTTTTTGGATCCATTC 3 ' 
-ACACCTTTTGTGGTTCTTCTTCGAAAATTCTAAAAAAAACCTGGGTAAGAA 
Y Ba/nHI 

3 ' CCTTTTGTGGTTCISINNNNISnSINNlSnSnsiAAACTAAAAAACCTAGGTAAG 5 ' 
-TGTGGAAAACACCAAGAAGCTTTTAAGATTTGATTTTTTGGACCCATTCTT 




-in by Pfu pol 
(then Dpnl) 



* 



BamHI 
then T4 ligase 




desired in-frame 
random mutants obtained 
following transformation 

Fig. 1 . Schematic representation of example primer design strategy for random mutagenesis 
of codons. Digestion at a unique restriction site followed by circularization with T4 DNA ligase 
excises a copy of the duplicated zone Y yielding desired in-frame mutants containing only X. 



4. Bacterial strains: In order to increase the transformation efficiency of large and ligated 
DNA libraries, the ultrahigh competent cells are strongly recommended. Escherichia coli 
DH10B™ (Life Technologies, Gaithersburg, MD) or Epicurian Coli®XL10-Gold 
(Stratagene) is ideal for production of larger primary libraries. 

3. Methods 

1. Design degenerate primers spanning amino acids targeted for mutagenesis, with random- 
ized codons encoded during primer synthesis. 

2. Run PCR on plasmids DNA using purified primers (see Note 6): 
95 °C initial denaturation, 5 min 

-* (95°C, 1 min -* 45°C, 1 min -» 68°C, 2 min/kb) 30 cycles 
-* 68°C extension, 10 min 
-» 4°C hold 



244 Kim and Guengerich 

3. Clean and concentrate PCR by QIAquick® PCR purification kit (Qiagen, Valencia, CA) 
and resuspend DNA. 

4. Digest cleaned DNA with the inserted restriction enzyme site (see Note 7). 

5. Gel-purify the digested PCR product using available "gene-clean" methods, e.g., 
QIAquick® Gel Extraction kit (Qiagen). Elute DNA in 300 uL distilled water or 10 mAf 
Tris-HCl , pH 8.5. 

6. Ligate DNA with T4 DNA ligase in a 350-u.L volume overnight at 16°C. 

7. Precipitate reaction mixture by ethanol/NaOAc, wash pellet twice with 75% ethanol at 
room temperature, dry, resuspend DNA in 22 uL water (11). 

8. Perform transformation using ultra competent cells such as E. coli DHlOb (Life Tech- 
nologies) or Epicurian Coli XL10 Gold (Stratagene) and recover in 300 \iL SOC medium 
(11, see Note 8). 

9. Plate pool on selective agar medium and incubate overnight at 37°C. 

10. Add 2-5 mL Luria-Bertani medium; scrape and shake cells into a thick suspension. 

11. Pool suspensions for each library and conduct alkaline lysis DNA preparation (11). 

4. Notes 

1. Only one of the primers (sense or antisense) needs to cover the target region in order to 
prevent self-hybridization, but both primers have to include any restriction enzyme sites. 

2. Although all of the 20 amino acids can not be introduced, PCR primer design according 
to Table 1 can eliminate the wild-type plasmid efficiently. In order to recover all 20 
amino acids codons in PCR, the codons in the target region can be encoded as 5'NNS3' 
(S = G or C in equal amounts) in the sense primer and 5'SNN3' in the corresponding 
region of the antisense primer. 

3. The total length of primers can be about 40-50 bp and can be extended up to 70 bp. 

4. The restriction enzyme site is incorporated into the primer by silent mutagenesis using 
the instruction provided in the New England Biolab catalog (Beverly, MA). 

5. The native pfu DNA polymerase is used for high PCR performance, but the conventional 
thermostable enzymes, such as Taq DNA polymerase, can be substituted when PCR con- 
ditions are optimized. 

6. The primer concentrations for PCR must be optimized by serial dilution (9). Because the 
primers are self-complementary, there is a strong tendency toward self-hybridization. 
Either too little or too much primer may result in reduced PCR yield. 

7. Additional digestion with the Dpnl, which cuts the dam-methylated parental DNA, may 
help to remove the contamination of the wild-type plasmids originating from the tem- 
plates in PCRs. 

8. High efficiency transformation depends highly on the use of ultrahigh-competent E. coli. 
Twenty successive transformations are conducted for the full production of the library. 

Acknowledgment 

The authors are grateful to Dr. A. Parikh for valuable suggestions and discussions. 

References 

1. Botstein, D. and Shortle, D. (1985) Strategies and application of in vitro mutagenesis. 
Science 229, 1193-1201. 

2. Hill, D. E., Oliphant, A. R., and Struhl, K. (1997) Mutagenesis with degenerate oligo- 
nucleotides: an efficient method for saturating a defined DNA region with base pair sub- 
stitutions. Meth. Enzymol. 155, 558-568. 



Random Mutagenesis 245 

3. Fromant, M., Blanquet, S., and Plateau, P. (1995) Direct random mutagenesis of gene- 
sized DNA fragments using polymerase chain reaction. Analyt. Biochem. 224, 347-353. 

4. Zhao, H., Giver, L., Shao, Z., Affholter, J. A., and Arnold, F. H. (1998) Molecular evolu- 
tion by staggered extension process (StEP) in vitro recombination. Nature Biotechnol. 16, 
258-261. 

5. Nakamaye, K. L. and Eckstein, F. (1986) Inhibition of restriction endonuc lease Neil cleav- 
age by phosphorothioate groups and its application to oligonucleotide-directed mutagen- 
esis. Nucl. Acids Res. 14, 9679-9698. 

6. Zaccolo, M., Williams, D. M., Brown, D. M., and Gherardi, E. (1996) An approach to 
random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleotide ana- 
logues. J. Mol. Biol. 255, 589-603. 

7. Tange, T., Taguchi, S., Kojima, S., Miura, K., and Momose, H. (1997) Improvement of a 
useful enzyme (substilisin BPN') by an experimental evolution system. Appl. Microbiol. 
Biotechnol. 41, 239-244. 

8. Zoller, M. J. (1992) New recombinant DNA technology for protein engineering. Curr. 
Opin. Biotechnol. 3, 348-354. 

9. Parikh, A. and Guengerich, F. P. (1998) Random mutagenesis by whole-plasmid PCR 
amplification. BioTechniques 24, 428-431. 

10. Lanio, T. and Jeltsch, A. (1998) PCR-Based random mutagenesis method using spiked 
oligonucleotides to randomize selected parts of a gene without any wild-type background. 
BioTechniques 25, 958-965. 

11. Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1989) Molecular Cloning. A Labora- 
tory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 



IV 

Cloning Unknown Neighboring DNA 



27 



PCR-Based Strategies to Clone Unknown DNA Regions 
from Known Foreign Integrants 

An Overview 

Eric Ka-Wai Hui, Po-Ching Wang, and Szecheng J. Lo 

1. Introduction 

Many foreign DNAs, such as some virus DNAs and almost all transposable ele- 
ments (transposons), are capable of integrating host genomes, and the effects of inte- 
gration can be pleiotropic. To investigate the mechanism and biological effect of 
foreign DNA insertions, characterization of the integration site, called integrant-host 
junction (IHJ), in the host genome becomes important. Traditional genomic library 
construction and screening for the cloning and analysis of IHJ are time-consuming, 
labor-intensive, and tedious. Therefore, a variety of efficient and reliable polymerase 
chain reaction (PCR)-based techniques have been developed. Application of the PCR 
to yield enough amounts of DNA for cloning and analysis is highly recommended 
especially for those specimens that are in a minute amount. Because the amplification 
process of PCR requires a pair of primers that can anneal to known sites at two end of 
the target DNA template, it seems that PCR is not applicable to IHJ searching because 
only one side of the fragment sequence in the integrant is known. A number of PCR- 
based techniques, however, have been developed to amplify the unknown cellular 
DNA flanking sequence from the foreign DNA. In this chapter, we introduce the PCR- 
based methodologies for the rapid acquisition of unknown DNA sequences. Based on 
the underlying principles, we classified these techniques into five categories: 1) PCR 
after intramolecular circularization; 2) interspersed repetitive sequence PCR (IRS- 
PCR); 3) ligation-anchored PCR (LA-PCR); 4) arbitrarily primed PCR (AP-PCR); 
and 5) reverse transcription PCR (RT-PCR). These techniques include inverse PCR 
(IPCR), partial IPCR (PI-PCR), long IPCR (LR-iPCR or LI-PCR), novel Alu-PCR, 
long interspersed repetitive element PCR (LINE-PCR), Bl-PCR, vectorette-PCR, 
multiplestep-touchdown vectorette-PCR (MTV-PCR), long-distance vectorette-PCR 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

249 



250 Hui, Wang, and Lo 

(LDV-PCR), splinkerette-PCR, thermal asymmetric interlaced PCR (TAIL-PCR), and 
retroviral LTR arbitrarily primed PCR (RELAP-PCR); and the capture PCR (C-PCR), 
which can improve the PCR amplification, is also discussed. 

2. PCR After Intramolecular Circularization 

A PCR technique, which is used to amplify an unknown DNA region adjacent to an 
integrated sequence after its intramolecular circularization, is called IPCR (inverse or 
inverted or inside-out PCR). 

2.1. IPCR 

The concept of IPCR first came in the 1980s (1-5) and has been in use for many 
years. The principle of this technique is illustrated in detailed in Fig. 1. IPCR begins 
with the digestion of genomic DNA with a restriction enzyme. Intramolecular self- 
ligation of restriction enzyme digested DNA fragments created a small monomeric 
circle. Within this circularized form of DNA, a conventional PCR technique is applied 
to amplify the IHJ region by using two opposite direction primers on the known 
integrant sequences called integrant specific primers (ISP). Hence, the primers 
are designed to anneal to the region of known sequence in IPCR. Generally, this tech- 
nique has been used to characterize fragments up to 5 kb (6). However, a new DNA 
sequence or restriction sites could be used to start a new round of IPCR to obtain 
additional information. This strategy may be repeated to "walk" both upstream and 
downstream of a known DNA sequence (7). IPCR has been already applied to identify 
the integration sites of hepatitis B virus (HBV) (8,9), human T-lymphotrophic virus 
type-1 (HTLV-1) (10-15), human immunodeficiency virus type-1 (HIV-1) (16), and 
some transposable elements such as IS30 (17), T-DNA, Ds element (18), dTphl (19), 
Tn5 (20), Tn55 (21), and P element (22). The insertion of reticuloendotheliosis virus 
(REV) on Marek's disease virus (MDV) genome is also identified by this method (23). 

2.2. PI-PCR and Long-Distance IPCR 

Some alternative IPCR methodologies have been published in recent years. 

Partial inverse PCR (PI-PCR) (see Fig. 2) employs the genomic DNA partially 
digested by using 4-base recognition restriction enzyme (such as SauiAl). After self- 
ligation, the circular DNA fragments are used as templates for IPCR (24). This is 
based on the preference of PCR for amplifying relatively smaller fragments. A DNA 
fragment that is less than 1 kb facilitates amplification via self-ligation and the IPCR 
process (9). A wide range of partial digests should be generated to find one that gives 
an optimal PCR amplification. Moreover, this approach eliminates the need to have 
any prior knowledge of restriction enzyme sites surrounding the integrant. 

Long-distance IPCR method, designated long-range IPCR (LR-iPCR) (25) or long 
inverse PCR (LI-PCR) (26), enables the direct amplification of relatively large size 
flanking from circularized DNA fragments. The central to the long-distance IPCR is 
the use of a thermostable polymerase. This technique has been adapted to amplify 
relatively large-size flanking fragments up to 10 kb by using a highly thermal stable 
polymerase. 



Strategies to Clone Unknown DNA Regions 



251 



Genomic 5'- 
DNA 3'" 



Restriction 
(T) enzyme 
digestion 



Unknown 




Known 


j= 


Unknown 


genomic DNA 




integrant 




genomic DNA 


region 


X 




—> 


region 


(left) 






(right) 



f f 

Edb oof 



T 



ISPl ISP3 

3'-* 5' ~m 



ISP2 ^ ISP4 ¥ 

RE(Jb ob e 

i 

T T 



t 

■a 



©Circulization by 
self-liaation 



self-ligation 



©Primer annealing: 
ISPl + ISP2 



(4) PCR reaction: ISPl + ISP2 





5' 

3' 

ISP2 5' 



I 5' ISPl 
I 3' 
I 5' 



Further amplification 

Agarose gel electrophoresis 

Southern hybridization 

Cloning 

Sequencing 

Data base alignment 



Fig. 1. Schematic flow diagram of IPCR protocol. Two complementary strands of genomic 
DNA have been shown at the top. The heavy and thin line regions represent the integrant frag- 
ment and cellular genomic unknown sequence, respectively. The positions of both left and 
right IHJs are indicated as closed circle and square signs, respectively. Integrant specific prim- 
ers ISPl, 2, 3, and 4 for 1-PCR are shown as arrowheads. In this particular example, the restric- 
tion enzyme cutting site (RE, scissors shape) is present in the integrant. ISPl and ISP2 have 
been applied to amplify the left IHJ. The right IHJ has been amplified by using the other pair of 
ISP, ISP3 and ISP4, under the same principle. If the RE is not present in the integrant, then any 
set of primer can be used to amplify both left and right IHJ (71). The slant lines on thick arrows 
indicate that no primer annealing will occur and no further amplification products. For detail 
manipulations see (9,17,97). 



252 



Hui, Wang, and Lo 







Unknown 

genomic DNA 

region (left) 


.2 

—3 
I 

Known 
W integrant 


—i 
I 

T 


Unknown 
genomic DNA 
region (right) 




Genomic 


1 

5' — 

3 | 


* 




II 

ISPl 
3'-* 5' 


ii 




l 

.3' 


DNA 


i 5'*- 3' 
T isp2 ' 


f 


j 


-b 1 
f 



RE4 



REIrJb RE2(Jd RE3(Jb 
(T) Restriction enzyme digestion 

T T 



RE5i 



RE3 



5' — I 
3 ■ 



res ;;;=■ 



RE2: 




®PCR using inverse primers: 
ISPl + ISP2 






-RE4 



RE5 



5— :, RE4 

5 



1 
@ Circulization by self-ligation 1 




Preference of 
PCR for amplifying 

Fig. 2. Description of PI-PCR. Two complementary strands of genomic DNA have been 
shown at the top. The heavy and thin line regions represent the integrant fragment and cellular 
genomic unknown sequence, respectively. The positions of both left and right IHJs are indi- 
cated as closed circle and square signs, respectively. The different 4-base restriction enzyme 
recognition sites (RE, scissors shape) have been marked from 1 to 5. The partial digested DNA 
fragments are self-ligated and can be amplified from a known ISP, ISP J and ISP2. PCR is 
preference to amplify smaller fragments even if there are comprable amounts of large and small 
DNAs. For detail manipulations see (24). 



Strategies to Clone Unknown DNA Regions 253 

2.3. Remarks for IPCR 

The major advantage of IPCR is to amplify the flanking unknown sequence by 
using two known specific primers on the integrant, so-called integrant specific primer 
(ISP). The intramolecular circularization (self-ligation) of template is a key step 
for IPCR (7). This technique, occasionally, does not produce any product in a particu- 
lar reaction, presumably because of the ineffective intramolecular ligation. This illus- 
trates that the circularization step in IPCR is a fastidious procedure and not easy to 
optimize. Wang et al. (9) have demonstrated that by choosing the nearest restriction 
site, which can be determined by conventional PCR, gives a higher successful rate for 
cloning the IHJ by IPCR. Moreover, the noncircularized, intermolecular ligation, and 
free viral or transposon DNA fragment may interfere with the PCR. If intermolecular 
ligation had occurred, multiple PCR products would have been generated. To avoid 
intermolecular ligation (ligation between two digested fragments), the concentration 
of DNA has to be decreased and this results in a large volume for ligation. In addition, 
IPCR has been proved to be less sensitive than the other PCR-based methods (//). 
Therefore, IPCR requires a relatively large size of sample for compensating the low 
efficiency of ligation. 

3. IRS-PCR-Based Techniques 

The principle of IRS-PCR (interspersed repetitive sequence PCR) is based on the 
fact that the IRS elements are interspersed in the human genome. In this technique, 
amplification proceeds with one ISP to the known integrant sequence and the other 
primer specific to the known cellular interspersed repetitive sequence (IRS), which is 
distributed among the genome. IRSs are present in a high copy number in most multi- 
cellular organisms (see Table 1) (reviewed in ref. 27). The extension products from 
these specific primers include a segment containing the region of IHJ. 

3.1. Novel Alu-PCR 

Novel Alu-PCR (novel Alu element-mediated PCR) is an IRS-PCR, which uses a 
primer to Alu element and ISP for the PCR amplification. The overall strategy of novel 
Alu-PCR is outlined in Fig. 3. Alu elements are the largest family of short interspersed 
repetitive elements (SINEs) (see Table 1). The average density of Alu repeats on 
human genome is at the mean interval of about 3-6 kb, although Alu is not uniformly 
distributed (reviewed in refs. 28-29). This technique actually was first applied to 
amplify human genomes in the background of nonhuman genome and called "Alu- 
PCR" (30-32). Hence, extending the applicability of Alu-PCR, the inserted foreign 
sequence can be directly amplified between the known inserted sequence and the Alu 
consensus sequence, and to identify the IHJ (33). 

Two specific primers are needed in novel Alu-PCR: ISP annealing to the known 
integrant sequence and the other to human Alu repeat sequences. In order to avoid 
illegitimate products, which are amplified from Alu sequences itself (Alu-Alu or inter- 
Alu amplification), two technical skills have been suggested (33). First, the primers 
should be synthesized by deoxyuridine triphosphates (dUTPs). This chemically modi- 
fied primer can then be destroyed by uracil DNA glycosylase (UDG) after the first 



Table 1 

Organization of the Human Genome 



DNA organization 



%of 

total genome 



Size of repeat unit 



Copy* 



IV) 



Human genome 

I. Mitochondrial genome 

II. Nuclear genome 

A. Genes and gene-related sequences 

1. Coding DNA 

2. Noncoding 

a. Introns, untranslated region, etc. 

b. Pseudogenes 

c. Gene fragments 

B. Extragenic DNA 

1. Unique or low copy no. 

2. Moderate to highly repetitive 

a. Tandemly repeated/clustered repeats 
i. Megasatellite DNA 

ii. Satellite DNA 
iii. Minisatellite DNA 
iv. Microsatellite DNA 

b. IRS approx 26.3 % 
i. SINE class 

- Alu family 

- MIR families 
ii. LINE class 

- LINE-1 (L1H or Kpn) family 

- LINE-2 family 
iii. LTR class 

- HERV/RTLV family 

- THE-1, MER, and other families 
iv. DNA transposon 

- mariner family 

- Others 
v. Others 



0.0005 % 
99.9995 % 
approx 25.0 % 
approx 2.5% 
approx 22.5% 



approx 75.0 % 
approx 45.0 % 
approx 30.0 % 
approx 3.7 % 



approx 8.7 % 
approx 7.0 % 
approx 1.7 % 
approx 10.6 % 
approx 8.5 % 
approx 2.1 % 
approx 4.6 % 
approx 1.3% 
approx 3.3% 
approx 1.6 % 



approx 0.8 % 



Full: 280 bp 
Average: 130 bp 

Full: 6 kb; Average: 0.8 kb 
Average: 250 bp 

Average: 1.3 kb 



Average: 250 bp 



approx 0.5-1 x 10 6 
approx 4 x 10 

approx 1-5 x 10 5 
approx 2.7 x 10 s 

approx 5 x 10 4 
approx 2 x 10 5 
approx 2 x 10 5 



approx 6 x 10 4 



HERV: human endogenous retroviruses; IRS: interspersed repetitive sequence; LINE: long interspersed nuclear element; LTR: long terminal 
repeat; MER: medium reiteration frequency; MIR: mammalian-wide interspersed repeat; RTLV: retrovirus-like elements; SINE: short interspersed 
nuclear element; THE-1: transposable human element. 



Strategies to Clone Unknown DNA Regions 



255 



Genomic 5' i 
DNA 3' I 



•-n Asymmetric PCR: 



IHJ 
Known m 
integrant 1 



Unknown 
genomic DNA region 



low cone, of 
Alu-Tag primer 
(synthesized by dUTPs) 5' 



5' ^-3' high 
^~ cone. 

»»- ISP1 



ISP1 + Alu-Tag primer 



Alu-Tag primer 
(synthesized 
by dUTPs) 5' 



Tag-Alu primer 
(synthesized 
by dUTPs) 



© UDGdigestic 



I 5' 

II 3' 



5' ^3' 

ISP 2 



Tag 5' 
primer^ 3' 





©Nested or hemi-nested PCR: 
ISP2 + Tag primer 



I 5' I 
3' 



i 



Further amplification 

Agarose gel electrophoresis 

Southern hybridization 

Cloning 

Sequencing 

Data base alignment 



*/ 




Fig. 3. Schematic diagram of the novel Alu-PCR. Two complementary strands of genomic 
DNA have been shown at the top. The heavy and thin line regions represent the foreign integrant 
fragment and cellular genomic unknown sequence, respectively. The position of IHJ is indi- 
cated by closed circle. Closed arrow boxes represent Ahi elements on the human genome in 
different orientations. Primers for PCR, ISP1, ISP2, Alu-Tag, and Tag primers, are shown as 
arrowheads. The non-annealing Tag region on Alu-Tag primer is shown by curved thin line. 
Human genomic DNA was amplified by using the first set of primers: ISP1 and Alu-Tag primer 
(step 1). After an initial 10 cycles of PCR, the Alu-Tag primers on new synthesized DNA is 
destroyed by UDG (step 2). These digest PCR products are further amplified by using an inter- 
nal primer: 1RS2 and Tag primers (step 3; nested PCR). Only DNA product I, which still con- 
tains both ISP2 and Tag nested primer target sites, will be amplified further. The cross marks 
on thick arrows indicate that no primer annealing will occur and no further amplification prod- 
ucts. For detail manipulations see (34). 



256 Hui, Wang, and Lo 

10-15 cycles of amplification. Such modification can break the Alu-Alu specific ampli- 
fication (see Fig. 3, step 2). Second, an asymmetric amplification (unequal ratio of 
two primers) is performed before UDG treatment (see Fig. 3, step 1). The primer on 
the known integrant sequence is added at least 10-fold higher concentration than the 
primer for the Alu sequence (34). In general, during the first 10-20 cycles, dsDNA 
products are generated. But when the limiting primer is exhausted, ssDNA is produced 
for the next cycles by primer extension (35,36). No matter the accumulation of dsDNA 
and ssDNA, the products of integrant-A/w amplification are higher than the Alu-Alu 
products, and thus this asymmetric PCR does not favor Alu-Alu amplification. 

In addition, the design of the primer contains a tag sequence, which can be applied to 
the other standard PCR protocols such as the nested or hemi-nested PCR (see Fig. 3, 
step 3), to decrease the nonspecific amplification of PCR. Moreover, a single primer 
control can exclude the false-positive amplification and Southern hybridization has been 
suggested to facilitate cloning. Some investigators, by using novel A/m-PCR, have success- 
fully identified cellular sequences flanking from integrated HBV (34), HIV-1 (37,38), 
and human papillomavirus type- 16 (HPV-16) (39) DNA. The adeno-associated virus 
(AAV) vector insertion for gene therapy, in addition, was also detected by this way (40). 

3.2. B1-PCR and UNE-PCR 

The Alu repeat is primate-specific but other mammals have similar types of 
sequence such as the B 1 family in mouse. Thus, the novel A/m-PCR equivalent, B 1-PCR 
has been applied to find the AAV vector integration site from the rat tissues (40). 

Based on the same principles, the Alu primer can be replaced by the others primers, 
which can anneal to the other genomic repetitive sequences. In addition to the SINE 
(such as Alu sequence) applied in novel-Alu PCR, other IRS has also been used under 
the same approach such as long interspersed nuclear element (LINE), and so-called 
LINE-PCR. LINE-PCR has been applied to identify the HPV integration site (39). 

3.3. Remarks for IRS-PCR 

The IRS-PCR offers at least four advantages over IPCR. First, less amount of DNA 
is required. Second, in contrast to IPCR, an intramolecular ligation reaction is not 
required in the IRS-PCR. This can overcome the low efficiency of the self-ligation 
reaction. Third, IRS-PCR is based on only two steps: UDG digestion and conventional 
PCR procedures, thereby saving most time. Fourth, IRS-PCR avoids the attendant 
problems in interpretation resulting from episomal contamination. 

However, this technique is not suitable for the case of IRS elements within a short 
distance to the integrant. Unfortunately, many virus genomes tend to insert adjacent to 
or into repetitive sequence, such as HBV (41-46), simian virus 40 (SV40) (47), murine 
leukemia virus (MuLV) (48), hamster endogenous retrovirus (49), HIV-1 (16,50-51), 
HPV-16 (52-54), woodchuck hepatitis virus (WHV) (55), and duck hepatitis B virus 
(DHBV) (56). Furthermore, the use of IRS-PCR is also limited by the requirement for 
the adjacent repeat sequences to be in the correct orientation. 

4. LA-PCR-Based Techniques 

LA-PCR (ligation-anchored PCR or cassette ligation-anchored PCR) is based on 
the ligation of an oligonucleotide cassette unit, called adapter or linker, to the cleaved 



Strategies to Clone Unknown DNA Regions 257 

genomic fragments (57). The amplification proceeds with ISP to the known integrant 
sequence and the other primer specific to the known ligated adapter. Indeed, the prin- 
ciple of single-specific-primer PCR (SSP-PCR) (58), rapid amplification of cDNA 
ends (RACEs) (reviewed in ref. 59), and rapid amplification of genomic DNA ends 
(RAGE) (60) have been developed using the similar concept. In these PCR methods, 
the ligated unit enables PCR to amplify the DNA fragment between itself and a known 
primer from known integrant sequence. 

4.1.LM-PCR 

In ligation mediated-PCR (LM-PCR), genomic DNA is digested by a restriction 
enzyme and ligated with a primer using T4 DNA ligase. Then, ISP and the ligated 
primer are used in a classical PCR amplification. This protocol has been applied to the 
samples from HTLV-I integration genome (11,61-62). 

4.2. Vectorette-PCR 

The unique feature of vectorette-PCR method is the special secondary structure of 
the cassette, which termed vectorette unit (see Fig. 4A). The vectorette unit contains a 
central non-complementarity mismatched region resulting in a bubble-shape (see 
Fig. 4A), therefore, the vectorette-PCR is also termed "bubble PCR." VectorettePCR 
was first used for rapid isolation of terminal sequences from yeast artificial chromo- 
some (YAC) clones (63), and then applied to the intronic DNA sequence characteriza- 
tion (64). The procedure of vectorette-PCR begins with the digestion of genomic DNA 
with a restriction enzyme to generate a 5'-overhang, and then ligation with a vectorette 
unit. The flanking sequences are then amplified by using an ISP of the integrant and 
the universal vectorette specific primer. The amplification strategy is summarized in 
Fig. 4B. The vectorette primer, which applied to the PCR is, actually, identity sequence 
to, but not complementary to, the noncomplementarity mismatched region of vectorette 
unit and therefore it only process PCR extention from the second round of the reac- 
tion. This enhances the PCR amplification specific to the IHJ containing genomic 
fragment. The HIV integration site has been identified by this method (16). 

4.3. MTV- and LDV-PCRs 

Vectorette-PCR, in addition, has been modified to be a multistep-touchdown 
vectorette-PCR (MTV-PCR) (65), which is suitable for analysis of the high CG con- 
tent region. MTV-PCR starts at a hot-start technique and proceeds at touchdown PCR 
cycle profile. Because the high GC content DNA results in the PCR conflicting sec- 
ondary structures, the application of touchdown cycling parameters prevent signifi- 
cantly the formation of unspecific DNA fragments. 

A vectorette-based long-distance PCR has been developed to amplify the fragment 
up to 5 kb (66), and so-called long-distance vectorette-PCR (LDV-PCR). The use of a 
mixture of thermostable DNA polymerases is central to this approach. 

4.4. Splinkerette-PCR 

However, undesirable amplifications of nonspecific "end-repair priming" may 
involve the free cohesive ends of unligated free vectorettes and 5'-overhangs of unknown 
cellular region (see Fig. 4C). These ends are filled during the first cycle of PCR. After 
the denaturing step, these ends are able to anneal together (as shown in step 4 of 



258 



Hui, Wang, and Lo 



Vectorette unit 



Mismatch 
region 




Vectorette 
primer 



Same sequence 
(not complementary) 



Splinkerette unit 



Mismatch 
, region , 



3' -*5' -^ 

Splinkerette 
primer 



Same sequence 
(not complementary) 




Furl tier amplification 

Agarose gel else! rap hop 

Cloning 

Sequencing 

Data base alignment 



No extension 





Further PCR cycle 
Nonspecific products 



Jl 



1st cycle of PCR: Filled in 



5'GATC ' / 

3' CTAG / 



Fig. 4. (A, facing page) Structure of the vectorette and splinkerette units. The primer for 
vectorette and splinkerette are also shown. (B, facing page) Schematic representation of the prin- 
ciple of vectorette-PCR. Two complementary strands of DNA are shown at the top. The heavy and 
thin line regions represent the foreign integrant fragment and cellular genomic unknown sequence, 
respectively. The position of IHJ is indicated by closed circle. ISP and vectorette primers for PCR 
are shown as arrowheads. The slant lines on thick arrows indicate that no primer annealing will 
occur and no further amplification products. For detail manipulations see (63). (C) A diagram show- 
ing the effect of "end-repair priming" in vectorette-PCR. (D) The splinkerette unit do not has "end- 
repair priming" effect. 



260 Hui, Wang, and Lo 

Fig. 4C). In this procedure, the complementary strand of vectorette primer is generated 
from unwanted fragments, and this decrease the specificity of vectorettes-PCR. The 
splinkerette is therefore designed as a hairpin structure on one strand rather than a cen- 
tral DNA mismatch (67), as compared in Fig. 4A. The advantage of splinkerette-PCR 
over vectorette-PCR is the elimination of the end-repair priming phenomena (see 
Fig. 4D). Some researchers have successfully identified flanking regions of transposon 
Sleeping Beauty (Tel I mariner superfamily) by using this method (68). 

4.5. Remarks for LA-PCR 

LM-PCR has been proved to be more sensitive than IPCR in the detection of 
integrant (11). Some commercial products (Invitrogen and TaKaRa), which use PCR 
technology to quickly identify the unknown sequence, are also based on the principle 
for LA-PCR. However, it requires a proper ligation between oligomer linker to 
genomic DNA fragments. The ratio of linker DNA and genomic DNA has to be seri- 
ally diluted to obtain a maximum intermolecular ligation (58). 

5. AP-PCR-Based Techniques 

The principle of AP-PCR (arbitrarily primed PCR) is using nonspecific arbitrary 
primers for PCR amplification (69). Following this "hemispecific" concept, the tar- 
geted gene walking PCR (70; reviewed in 71), single primer reaction (72), differential 
display PCR (DD-PCR) (73; reviewed in 74,75), and restriction site PCR (RS-PCR) 
(76; reviewed in 71) have been developed for the amplification of DNA sequences by 
nonspecific arbitrary primers. 

5.1. TAIL-PCR 

TAIL-PCR utilizes three nested specific primers on known integrant in successive 
three rounds of PCRs together with a shorter arbitrary degenerate (AD) primer. The 
basis for this strategy is thermal asymmetric PCR. Arbitrary priming creates nontarget 
molecules, because degenerate primers that hybridize randomly in genomic DNA and 
constitute the bulk of the final unwanted products. The interspersing asymmetric and 
symmetric PCR cycles are used geometrically to favor amplification of target mol- 
ecules over nonspecific products. 

A schematic diagram of targeted TAIL-PCR is shown in Fig. 5. During the high- 
stringency cycle at first round of PCR (high-stringency PCR program at step 1 in 
Fig. 5) only the long integrant specific primer ISR 1 can efficiently anneal to the DNA 
template, therefore only specific product (product I in Fig. 5) is amplified and little or 
no nontarget sequence product (which is primed at both ends by AD primers; product 
II in Fig. 5) has been formed. In the following single reduced-stringency cycle (low- 
stringency PCR program at step 1 in Fig. 5), however, both ISR 1 and AD primers can 
anneal to the template DNA. The single-stranded target DNA, which is produced dur- 
ing last high- stringency cycles is replicated to dsDNA and hence providing a several- 
fold increase of target template for the next round of amplification. In following 
TAIL-cycling (TAIL programme at step 1 in Fig. 5), the specific product (product I) is 



Strategies to Clone Unknown DNA Regions 



261 







Known 


T 


Unknown 










integrant 


genomic DNA region 


-\__ 


3'<15' 




I 




H 




3'<5' 

: 3'<5' 


Genomic 










-3' 
-5' 


short 


DNA 













(?) Primary PCR reaction: ispi + primer AD 



Stringency: 
Cycle #: 

92°C 
72°C 
62°C 



High 
5 



■=>' 



TAIL 
12-15 



"Lr- 


"Ir 


*r 



DAPDAPDAP 



f Product I: Specific product 
(from ispi + ad) 
Yield: Moderate 
Detection (agarose gel): +/- 



ISP2 ISP3 



AD primer 

<! 

3' 
5' 



Product II: Non-specific product 

(from ad primer) 
Yield: Low 
Detection: - 



AD primer 



> 

AD primer 



Yield: High 

Detection: 



: Non-specific product 

(from ispi) 



©1000X dilution 
Secondary PCR reaction: ISP2 + ad primer 



o^r, T Stringency: TAIL 

PCR programme L Cyc| ^ #; 1(y]2 



Product I: Specific product 

(from ISP2 + AD primer) 

Yield: High 

Detection (agarose gel): ++ 



Product II: Non-specific product 

(from AD primer) 
Yield: Very low 
Detection: - 




AD primer 



AD primer 

< 

3' 

5' 



© 



1000X dilution 

Tertiary PCR reaction: ISP3 + AD primer 



PCR programme [ gW ™L 



Product I: Specific product 

(from ISP3 + AD primer) 
Yield: High 
Detection (agarose gel): ++ 



Product II: Non-specific product 

(from AD primer) 
Yield: Very low 
Detection: - 



Fig. 5. Summary of the TAIL-PCR procedure. Two complementary strands of DNA have 
been shown at the top. The heavy and thin line region represent the integrant fragment and 
cellular genomic unknown sequence, respectively. The position of IHJ is indicated by closed 
circle. ISP and AD (arbitrary degenerate) primers for PCR are shown as close and open arrow- 
heads, respectively. The PCR program with different stringencies and cycle number is shown 
on the top of the box. D, A, and P represents denature, annealing, and polymerization step, 
respectively, in PCR cycle. For detail manipulation see (84). 



262 Hui, Wang, and Lo 

possible to be amplified preferentially over nontarget sequence product. But at the 
same time, nonspecific product (at both ends by the long ISP I primers; product III in 
Fig. 5) can also arise efficiently through mispriming. Such undesired products are 
diluted out, however, in subsequent secondary (step 2) and tertiary (step 3) round 
PCR, which using internally nested specific primers ISP2 and ISP3, respectively. The 
TAIL cycling in both secondary and tertiary is performed in lower background fur- 
ther. In fact, the T-DNA insertion (77,78), Ds elements (79,80), and Ttol introduced 
(81) in Arabidopsis have been identified by this technique. 

5.2. RELAP-PCR 

The combination of a long ISP designed to detect retroviral long terminal repeat 
(LTR) and a short arbitrary primer (AD primer) from the AD primer set in different 
lengths binding in a random fashion under a low-stringency condition are used (see 
Fig. 6, upper panel). Therefore, AP-PCR had been adapted to allow the amplification 
of LTR-containing retrovirus integration site. This is called RELAP-PCR (retroviral 
LTR-arbitrarily primed PCR) and has been applied to identify the integration site of 
mouse mammary tumor virus (MMTV) (82). 

Hot spot-combined PCR (HS-cPCR), modified AP-PCR for retrovirus, is based on 
previous finding regarding the host spot of retroviral LTR integration sites. In this 
technique, the primers have been designed to target on both known retroviral LTR and 
nonintegrant region in different combination (83). It is possible to design primers, 
which border the "suspected" fragment. 

5.3. Remarks for AP-PCR 

These AP-PCR methods allow rapid detection without any DNA manipulation 
before PCR, such as restriction enzyme digestion or ligation. Amplification occurs 
either upstream or downstream from a known sequence. In TAIL-PCR, a set of nested 
long primer and a short arbitrary primer are important (77,84). Besides the primer 
design, the stringency in the primer-template interaction is an important parameter of 
this class of PCR (85). Specificity of the amplification reaction has been further con- 
firmed by Southern blotting of the PCR products. The single primer control is always 
necessary and important to exclude the false positive results. 

In the case of RELAP-PCR, a series of walking primers have been designed to 
increase the incidence of positive results. In addition, a series of walking reaction are 
usually done in parallel. This can be laborious and time-consuming. Moreover, this 
strategy is only suitable for the LTR-containing integrant. 

6. RT-PCR-Based Technique 

In the case of retrovirus, the promoter activation within 3' untranslated LTR ini- 
tiates chimeric mRNA transcripts, which consist the viral LTR and the cellular gene 
fragment in the same transcriptional orientation (86,87). Based on this property of 
retrovirus, the poly(A)-tail containing mRNA is purified and cDNA is synthesized by 
reverse transcription using an oligo(dT)-adaptor primer which primers on the poly(A) 



Strategies to Clone Unknown DNA Regions 



263 



/^RELAP-PCR 



LTR specific 
primer 



(right) 

T 



LTR (left) 




Single- 
spliced: 
Double- 
.spliced: 



Chimeric mRNA: HIV minor transcript 



RT-PCR 

RT: 



-.PCR: 



3'TTTTTTT^' (iT)-adapU 

primer {ONA) 



Further amplification 

Agarose gel electrophoresis 

Southern hybridization 

Cloning 

Sequencing 

Data base alignment 



Fig. 6. Summary of the procedure for RELAP- and RT-PCR in the studying on retrovirus 
insertion. Two complementary strands of HIV DNA have been shown at the middle. The heavy 
and thin line region represent the integrated HIV fragment and cellular genomic unknown 
sequence, respectively. The position of left and right IHJ is indicated by closed circle and 
square, respectively. Boxes with HIV open reading frames (ORFs) are shown. The right and 
left LTR region of HIV are shown as an open box (the size is not to scale). Primers for PCR are 
shown as arrowheads. Upper panel is the schematic diagram for RELAY-PCR. For detail 
manipulation for see (82). Lower panel is the schematic flow diagram for RT-PCR based pro- 
tocol. The major and minor viral transcripts from HIV viral transcription are shown. Only the 
cDNA from chimeric mRNA contains LTR primer target site. For detail manipulation see (90). 



tails. Using the adapter primer and an LTR-specific primer, the chimeric mRNA con- 
taining the retrovial insertion site is amplified by PCR. The overall RT-PCR based 
method to isolate these chimeric cDNAs is schematically shown in the lower panel in 
Fig. 6. This principle is similar as anchored PCR (A-PCR) (88), one-sided PCR (89), 
and RACE. This RT-PCR based method is rapid and simple, and successful to identify 
the retrovirus integration site (90). However, this method only works on the virus, 



264 



Hui, Wang, and Lo 



AD-Tag 
5' primer 



AD-Tag 
5' primer 

V>3 




LTR2 A 
3'-««i «©5' 



Genomic 5' 
DNA 3' ■ 



IHjl 

(left) - 



LTR (left) 



3' 5' 






Tag ^3^ 
primer 



LTR2 




LT.R2 XI 



Unknown 

genomic 

DNA region 



m 



HIV genome 



Q} PCR reaction: biotinylated LTR1 + AD-Tag primer H 

^ Capture: streptavidin bead (isolation of biotinylated DNA) II 



3' 5' 

f a ^ -a ^"™ 



Tag ^3. 
primer 



@ PCR reaction: biotinylated Tag primer + LTR2 

@ Capture: streptavidin bead (isolation of biotinylated DNA) 1 



*: 



t (right) 1 



IHJ 

(right) 



O 



Direct sequencing 
Data base alignment 



which contains a cis-acting promoter activity sequence (like LTR) and synthesizes the 
chimeric mRNA. 

7. Capture PCR Improvement 

The capture PCR (C-PCR) is an alternative protocol to enrich the interested DNA 
fragments by a streptavidin-coated support for the PCR (91). Indeed, both AP- and 
LM-PCR have been improved by this protocol. 

Under the concept of AP-PCR, the biotinylated integrant specific primer and a 
partly degenerate arbitrary primer are applied for the PCR. The amplified DNA frag- 
ment is then isolated by streptavidin-coated magnetic beads (92). The application of 
this approach into AP-PCR is shown in Fig. 7A. This method has been used for the 
isolation of the integrated retroviral provirus (92). Under the concept of LM-PCR, 
after initial ligation of oligonucleotide adapter to all restriction ends, the biotinylated 
specific primers to known sequence are used for an extension reaction. These biotin- 
labeled extension products are immobilized on streptavidin-coated beads and then used 
as templates in a PCR. This technique is also called amplification of insertion 
mutagenised sites (AIMS) (93). The application of this approach into LM-PCR is 
shown in Fig. 7B. This method has been applied to the detection of Bx\ gene in maize 
by transposon tagging Mutator (93,94). This improvement of alternative AP-PCR is 



Strategies to Clone Unknown DNA Regions 265 



IHJ 

Unknown 



r\nown ^ unmiuwm 

D integrant T genomic DNA region 




biotinylated ISP1 



©Extension: J^ 1 

biotinylated ISP1 <SL ^- 

J 

©Capture: i 

streptavidin bead (solid phase) ^ 

• priM 



5' 
Q3' 



*»-3' 

[32PJISP2 



(5) PCR: 1 

^ [32p]jsP2 + linker primer My 

T 



— , 3 , Gel electrophoresis 

w ^^ ,— , g-k Elution 

^^ Sequencing 

Data base alignment 

Fig. 7. Outline of application for the C-PCR into AP- or LA-PCR. Two complementary 
strands of DNA have been shown at the top. The heavy and thin line regions represent the 
integrant fragment and cellular genomic unknown sequence, respectively. The position of IHJ 
is indicated by closed circle or square. (A, facing page) Principle for C-PCR modification in 
AP-PCR. For detail manipulation see (92). (B) Principle for C-PCR modification in LA-PCR. 
For detail manipulations see (93). 



simple and highly specific. Moreover, no cloning procedure is required if solid-phase 
sequencing is used. 

8. Discussion 

There is much interest to characterize the unknown neighboring DNA from a pre- 
sumed integration site. The identification of the IHJ is important not only for an under- 
standing of the molecular mechanism of integration, but also for identifying novel 



266 Hui, Wang, and Lo 

cellular genes that are involved in cell proliferation and differentiation (95,96). How- 
ever, genomic cloning method requires the establishment of genomic DNA libraries, 
which is time-consuming and laborious. Therefore, many PCR-based techniques have 
been developed for the elucidation of unknown flanking DNA sequence adjacent to a 
region of known integrant sequence. The genomic sequences flanking foreign integrant 
can then be determined rapidly with these techniques (within 1 wk). PCR-based tech- 
niques offer an inexpensive and flexible alternative to IHJ searching, and can be per- 
formed in any laboratory equipped with basic molecular biology. The limitation of 
PCR is the need for the sequence of two target specific primers that flank the region 
that is intended for amplification. The problem here is how to allow the direct ampli- 
fication of DNA without a prior knowledge of sequence information. Several strate- 
gies have resolved this limitation. IPCR can amplify flanking region directed away 
from the core region of known integrant sequence after DNA self-ligation (circular- 
ization). IRS-PCR depends on the distribution of IRS on the genome. Many tech- 
niques for amplifying flanking unknown regions of DNA are based on the creation of 
new primer binding sites on the potential PCR template by ligating oligonucleotide 
linkers or cassette unit of known sequences to the ends of DNA fragments, such as 
LA-PCR. And some other techniques allow primer binding in a random fashion under 
low-stringency condition, such as AP-PCR. All methods described in this chapter are 
compared in Table 2. 

Beside the IHJ searching, these techniques can also be applied to the determina- 
tion for YAC end points (31,63), cDNA ends (97), genomic breakpoints (deletion or 
translocation) (66,98), intron-exon junctions (99), gene rearrangements (6), promoter 
sequence sequences (100,101), mating-type gene switching (102), and gene-target- 
ing vector construction (103). Although, some PCR-based techniques such as RAGE 
(104), RS-PCR (76; reviewed in 71), panhandle PCR (105,106; reviewed in 107), 
multiplex RS-PCR (108), gene walking PCR (109), homo-oligomeric tailing based 
PCR (110), novel step-down PCR (111), and nonspecifically primed suppression 
PCR (NSPS-PCR) (112) have not yet been used to IHJ study, these principles are 
also applicable in the integration site searching. 

Among all techniques introduced in this chapter, the IRS-PCR- and TAIL-PCR- 
based methods are highly recommended to apply in the integrant seeking. One reason 
is the high sensitivity of these two methods. Second, genomic DNA manipulation, 
such as DNA digestion and ligation reaction, is not required. Third, only the simple 
straightforward technique of conventional PCR protocol is needed. In fact, each of 
the approaches to identify IHJ is useful, and the experimental context is the critical 
feature that determines success. If an experiment is poorly designed (especially the 
primer set sequence) or the sample is contaminated, the result is a large number of 
bands after PCR, that are difficult and time-consuming to analyze. Any inefficiencies, 
mispriming, or incomplete reaction in the PCR or restriction enzyme digestion steps 
can result in artifacts that are misleading. Some improvements, therefore, are applied 
in the PCR reaction to increase the specificity of PCR amplification. These are hot 
start PCR (in any PCR techniques), nested PCR (in IRS-PCR, TAIL-PCR), touch- 



Table 2 

Methods Described in this Chapter 

















Source of 




Principle 




DNA 


DNA 


Amount of 






nonspecific 




Base on 


Methods 


digestion 


ligation 


DNA used 


Primers on 


Step to optimize 


products 


Sensitivity 


Inverse 


IPCR (Fig. 1) 


+ 


+ 


D: 0.2-10 ug 


Integrant 


1. Relative high DNA 


1. Unintegrated 


Low 




PI-PCR (Fig. 2) 




(self- 


L: 0.2-5 ng/uL 




sample amount 


DNA 






LR-iPCR 




ligation) 






2. DNA concentration 


2. Unligated 






LI-PCR 










of self-ligation 


DNA 




IRS-PCR 


Novel Alu-?CR 
(Fig. 3) 
LINE-PCR 
Bl-PCR 






P: 10-106 ng 


1. Integrant 

2. IRS (eg. Alu, 
LINE, Bl) 


1. IRS orientation 

2. Distance to IRS 


Inter-IRS 
amplification 


High 


LA-PCR 


LM-PCR 


+ 


+ 


D: 0.5-2 ug 


1. Integrant 


1 . Ligation efficiency 


End-repair 


Medium 




Vectorette- 






L: 0.5-2 ug 


2. Adapter unit 


2. Adapter unit design 


priming 






PCR (Fig. 4B) 










(Fig. 4C) 








MTV-PCR 


















LDV-PCR 


















Splinkerette- 


















PCR 


















Capture (Fig. 7B) 
















AP-PCR 


TAIL-PCR 
(Fig. 5) 
RELAP-PCR 
(Fig. 6) 
Capture (Fig. 7A) 






P: 20-150 ng 


1. Integrant 

2. Arbitrary 
primer 


Primer set design 


Arbitrary 
priming 


High 


RT-PCR 


RT-PCR (Fig. 6) 




+ 


R: 3 ng poly(A) + 
RNA 


l.LTR (integrant) 
2. Adapter 


cDNA synthesis 




Medium 



D: DNA digestion reaction; L: ligation reaction; P: PCR reaction; R: RT reaction. 



268 Hui, Wang, and Lo 

down PCR (in IRS-PCR, MTV-PCR, and TAIL-PCR), specific primer cassette struc- 
ture (in vectorette- and splinkerette-PCR), asymmetric (or unequal) ratio of the two 
amplification primers is used (in IRS-PCR and TAIL-PCR), primer synthesis by dUTP 
and then digest by UDG (in IRS-PCR), isolated biotinylated products (in C-PCR), and 
-NH2 and -P04 groups modification on the adaptors to prevent nonspecific 3' end 
elongation during PCR reaction (in novel step down PCR). These modifications are 
pivotal to successful amplification. It might also be helpful to optimize the PCR con- 
ditions with respect to magnesium and dimethyl-sulfoxide (DMSO) concentrations 
according to standard protocols. Although false positive results could happen, both 
single primer control and Southern blotting would minimize this problem and confirm 
the specificity of the amplified products. 

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28 

Long Distance Vectorette PCR (LDV PCR) 
James A. L. Fenton, Guy Pratt, and Gareth J. Morgan 

1. Introduction 

Vectorette polymerase chain reaction (PCR) is a method designed to amplify DNA 
when the sequence of one end of the target DNA is unknown (1,2). This technique, 
therefore, gives a handle on unknown sequence, which flanks DNA that has already 
been characterized, or sequenced. The vectorette method was conceived and patented 
in 1988 when it was used to sequence the termini of YAC clone inserts (7), as well as 
to undertake genomic walking (2). Other applications have been developed, including 
sequencing of cosmid insert termini, mapping of promoters, and/or introns in genomic 
DNA from cDNA subclones, sequencing of large clones without subcloning, mapping 
of regions containing deletions, insertions, and translocations. Vectorette PCR has 
also been adapted to clone full-length cDNA and determine the 5' and 3' ends of 
mRNAs (3). 

Vectorette PCR has been utilized for the amplification and sequencing of genomic 
breakpoints in translocations in chronic myeloid leukemia (CML) (4,5). These 
breakpoints were isolated using relatively small PCR fragments across the breakpoint 
of a known translocation, t(9;22) (q34;l 1), which produces the chimaera gene BCR- 
ABL. More recently, we have developed and applied a robust long-distance vectorette 
(LDV PCR) PCR strategy, which was initially developed to isolate the specific sites of 
DNA breakpoints in chromosomal translocations and recombination events in the 
hematological malignancy multiple myeloma (6). Unlike the previous studies where a 
known translocation was being analyzed, the aim was to effectively screen patient 
DNA for unknown translocation and recombination events. The most obvious require- 
ment for such a method is that longer PCR products would have to be obtained from 
vectorette PCR. To this end, the protocol for LDV PCR was developed. 

The vectorette unit (see Fig. 1) is an oligonucleotide linker of synthetic double- 
stranded DNA, which possesses a restriction fragment-compatible end that can be 
ligated to. In fact, a vectorette unit is only partially double stranded because there is a 
central mismatched region (see Fig. 1). This mismatch region was incorporated as part 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

275 



276 Fenton, Pratt, and Morgan 

Mismatch region 




■5' 



^ / 



SP Lower strand 



■*- 



VP 



"* nVP 

Vectorette primer(s) 

Fig. 1. Schematic representation of a vectorette unit. The vectorette unit is partially double 
stranded and contains a central mismatched region to avoid first strand synthesis by the 
vectorette primers. Two primers can be utilized for two rounds of PCR, vectorette primer (VP) 
and the nested vectorette primer (nVP). There are also sequencing primers (SP), which bind 
internally to the PCR primers, SP can be used to directly sequence any PCR products obtained. 



of a strategy to avoid non-specific amplification, which can commonly occur in one- 
sided PCR techniques. Vectorette PCR consists of three basic stages: 

1. Restriction digest of the sample DNA, this usually generates a 5' overhang (see Fig. 2) 

2. Ligation of a compatible vectorette unit to the restriction enzyme-digested end (see Fig. 2) 

3. PCR using primers from the known DNA sequence (called the initiating primer, or IP) 
and primers for the vectorette unit (vectorette primer, or VP) (see Fig. 3) 

The importance of the central mismatch region in vectorette units is that it is this 
part of the design of the vectorette unit, which negates any nonspecific priming 
occurred. The vectorette primer has the same sequence as the bottom strand of the 
mismatched region and thus is unable to bind to this sequence, i.e., anneal to the 
vectorette DNA during the first cycle of PCR. Therefore, only the IP, complimentary 
to the known part of the sequence of interest, will anneal in the first cycle of PCR and 
therefore prime DNA synthesis (see Fig. 3). During the first cycle of PCR, the priming 
form the IP will eventually produce a sequence complimentary to the bottom strand of 
the vectorette unit. This now provides a template to which the VP can bind and, thus, 
prime, which means that consequent rounds of PCR (from cycle 2 onward) proceed as 
conventional PCR, so that there is only amplification of fragments containing the 
sequence of interest and ligated vectorette units. Our experience has shown that in 
order for a successful LDV PCR protocol to work, a nested PCR is also required. 

The type of sequence to be investigated will determine what is observed when 
LDV PCR products are run out on an agarose gel. If one is simply trying to charac- 
terize unknown sequence adjacent to known DNA, then any band obtained will be 
potentially interesting. However, if studying recombination events (including trans- 
locations) in a specific sequence region where the germline configuration is already 



LDVPCR 

R 



R 



S 



R 



R R 



R 



R 



Known sequence 



R 



R 



S 



R 



R 



R R 



277 



Genomic DNA 



digest with R 



ligate vectorette 
units ( ^H) 
I to digestedDNA 



Vectorette library 



Fig. 2. Formation of vectorette library. Genomic DNA is digested with a Restriction enzyme 
(R) for 1 h. Vectorette units (dark rectangles) are ligated to the now compatible ends of 
the restriction digested DNA, giving the vectorette library, which will include fragments with 
the known DNA of interest. 



known and germline DNA is known to present within a sample, then more caution is 
required. When an appropriate restriction site is within range, LDV PCR will amplify 
a band of predictable size from any germline DNA present, and in informative cases, 
a band of different size from DNA template that has been subject to recombination 
(see Fig. 4). Of course, amplifying germline bands does provide suitable positive 
controls for the technique. 

Reviewers of inverse PCR methodology have reported that vectorette PCR can give 
spurious amplification of nontarget DNA (7,8). Such undesirable amplification of non- 
specific "end-repair priming" may involve the free cohesive ends of unligated free 
vectorette units and 5' overhangs of unknown cellular regions (8). In both cases, the 
ends are filled during the first cycle of PCR and are thus able to anneal together after 
the subsequent denaturation step. A complimentary strand for vectorette primer is now 
in position for PCR to be initiated from this nonspecific fragment. As a rule, we have 
not encountered major problems with such nonspecific priming, this may be a result of 
the hot start initiation steps, which has been incorporated into the protocol. 

One of the advantages of vectorette PCR, in our hands, is that we have found it to 
be a simpler and more rapid assay to perform than Southern blotting, which may oth- 
erwise have been undertaken. LDV PCR allows the simultaneous detection and isola- 
tion of recombination breakpoint regions, i.e., the boundary between known sequences 
of DNA and unknown sequences. Additionally, the technique can be applied to quan- 
tities of DNA that are too low to permit realistic Southern blotting to take place. The 
amplification nature of the protocol described also allows one to isolate DNA recom- 
bination events such as a translocation in a small subpopulation of cells, against a 
background of nonrearranged (germline) DNA from other (normal) cells. 



278 Fenton, Pratt, and Morgan 

IP 1 st round of LD V PCR 

^ I 1__^^ initiates from primer IP 

^ ' ' from known sequence only 

vp » 2nd round of LDV PCR 

^^ I I ^ extension from IP and VP. 

^ ' ' ™ (VP only hybridizes to 

product of 1st round PCR) 



VP , IP 



n cycles of PCR 
between IP and VP 



Fig. 3. LDV PCR. The IP binds to the known squence (white rectangle) and primes DNA 
synthesis. If a restriction site with a ligated vectorette unit is within range, a sequence compli- 
mentary to the bottom strand of that vectorette unit will be produced. This provides a template 
to which VP can bind and thus prime allowing conventional PCR to occur from cycle 2 onward. 

Germline (A) Unknown (B) 

R __IP R ^_IP 



I 



LDV PCR 

(n cycles) 



■ (A) Predictable size 

(B) Unpredictable size 

Fig. 4. Schematic of LDV PCR products on agarose gel. When investigating a recombina- 
tion event, bands of predictable size (A) may be amplified from germline DNA in addition to a 
band of different size from DNA template, which has undergone a recombination event (B). 
The band (A) of predictable size acts as a useful control in this case. 

2. Materials 

2.1. DNA Extraction 

Genomic DNA extracted from the sample of interest. The DNA to be investigated 
must be digestible with an appropriate restriction enzyme, yielding a general popula- 
tion of DNA fragments (see Note 1). 

2.2. Construction of Vectorette Libraries 

1. Restriction enzymes. A range of restriction enyzmes is recommended for LDV PCR 
(see Note 2). 

2. Vectorette units. These are supplied commercially. There is a starter kit available, namely 
Vectorette II (Sigma-Genosys). 



LDVPCR 279 

3 . 1 00 mM Dithiothreitol (DTT) . 

4. 100 mM Adenosine triphosphate (ATP). 

5. T4 Ligase. 

2.3. Primers 

1. Vectorette and nested vectorette primers (Sigma-Genosys). 

2. Initiating primers (see Subheading 3.3.). 

2.4. LDVPCR 

1. Taq polymerase, a suitable "long accurate" polymerase enzyme should be employed, e.g., 
LA Taq (TaKaRa) (see Note 3). 

2. Thin-walled/ultrathin-walled PCR tubes. 

3. Agarose. 

4. Ethidium bromide. 

5. TBE buffer. 

2.5. Sequencing 

1 . QIAquick Gel Extraction kit (QIAGEN). 

2. Vectorette sequencing primer, a 15mer and 20mer are both available (Sigma-Genosys). 

3. ABI Prism Big dye terminator (Applied Biosystems). 

2.6. Cloning 

1. pT7Blue Vector Perfectly Blunt Cloning Kit (Novagen) 

3. Methods 

3.1. DNA Extraction 

"Good quality" DNA is required as a template. The DNA should be accurately 
quantified so exactly 1 jig is used in the construction of the Vectorette libraries 
(see Note 1). 

3.2. Construction of Vectorette Libraries 

3.2. 1. Restriction Digest 

For each vectorette library, 1 pig of DNA is required, and the number of vectorette 
libraries to be constructed should be decided (see Note 2). 

The appropriate amount of DNA solution is added to a 0.5 ml microfuge tube, 
along with the following reagents for a restriction digest: 

1. 1 [tg of genomic DNA. 

2. 5 [xL of 10X restriction buffer. 

3. 20 Units of restriction enzyme. 

4. Sterilized/deionized water to 50 [xL final volume. 

The sample is incubated in a heat block/water bath at 37°C for 1 h, and then placed 
on ice for at least 2 min. Once chilled, the restriction digested DNA is ready for the 
ligation step. 

3.2.2. Ligation 

Each vectorette unit is supplied in a vial as 15 pmol of annealed, lyophilized DNA. 
The contents of the vial should be resuspended in 25 [ih of sterilized/dionized water. 



280 Fenton, Pratt, and Morgan 

1. 5 uL of the corresponding vectorette unit is ligated to the digested DNA sample with the 
following cofactors: 

a. 5 (xL Vectorette unit, (now in solution). 

b. 1 uL 100 mM ATP. 

c. 1 uL lOOmMDTT. 

d. 1 fxL (1 Unit) T4 DNA ligase. 

2. The sample is incubated at 20 C C for 60 min, followed by 37°C for 30 min, both steps are 
repeated twice on a thermocycler (i.e., 4.5 h in total). The purpose of this cycling is to 
increase the efficiency of the ligation step (see Note 4). 

3. Add 200 uL of sterilized/dionized water to each tube and store the vectorette libraries in 
aliquots at -20°C. This provides the DNA template for the PCR. 

3.3. Primers 

Two lyophilized PCR primers, vectorette II primer and nested vectorette II primer, 
are commercially available (Sigma-Genosys). These should be resuspended in 1 mL 
of sterilized/dionized water to give a working concentration of 10 \iM. It is recom- 
mended that aliquots of these solutions are made up and stored at -20°C. 

IPs need to be designed for the known (anchor) end of the LDV PCR. This can 
easily be achieved by using a primer design program, such as Primer Express (Applied 
Biosystems) if there is enough known sequence available. Obviously, such programs 
will only design primer pairs, but this can be useful because it allows the opportunity 
to test out beforehand primers, which are going to be applied to LDV PCR. The prim- 
ers used must be totally specific for the known target sequences and be based on the 
"standard" design parameters of length (no. of basepairs), the GC/AT ratio and melt- 
ing temperature (T m ). Primers and nested primers are required to bind to the known 
sequence close to the boundary between the known and unknown DNA sequences. 
Seminested primers are just as acceptable, if there are constraints on space. Ideally, we 
prefer to use primers that have a high T m value so that a 2-step PCR protocol can be 
used (see Note 5). 

3.4. LDV PCR 

The following protocol describes the use of LA taq (TaKaRA, Japan), but this is 
not the only suitable DNA polymerase enzyme commercially available (see Notes 3). 

3.4. 1. First-Round PCR (30 cycles) 

1. For the 50-uL volume reaction, use thin-walled/ultrathin-walled PCR tubes appropriate 
to the thermal cycler, which will be employed. Set up each reaction by adding 8 uL dNTPs 
(2.5 mM each), 5 uL 10X LA PCR buffer (Mg plus), 0.5 uL Vectorette primer (10 (xM or 
10 pmol/uL), 0.5 u,L IP (10 \iM or 10 pmol/uL), 14 uL sterilized/deionized water. 

Always ensure everything is kept on ice at all times. These reagents can be made up 
together in master mix form and added 28 uL (see Note 6) to 2 uL DNA (vectorette 
library), giving a total volume of 30 uL. 

2. If necessary, overlay the reaction with mineral oil and briefly spin down the tubes. 

3. Place tubes on thermocycler and start a program with following cycling profile: Initial 
denaturation at 95°C for 3 min, followed by 30 cycles consisting of denaturation at 95°C 
for 45 s, annealing/extension at 68°C for 4 min. The reaction is completed with a hold for 
final extension at 72°C for 10 min and a final hold at 4 C C, until the tubes are removed. 



LDVPCR 281 

4. Employ a manual hot start (see Note 7). Ensure that there is a hold/pause during the initial 
3 min 95°C denaturation step. After at least a minute at this temperature, the contents of 
the tubes will effectively have equilibriated. Add 20 uL of sterilized/dionized water per 
tube, containing 0.5 uL of LA Tag (TaKaRa), through the mineral oil layer, giving a final 
total volume of 50 uL. It is easier to make up enough of this latter solution for the required 
number of tubes as a master mix beforehand. When all the enzyme has been added to 
every tube, restart the cycle program. 

3.4.2. Second-Round (Nested) PCR (35 cycles) 
A nested PCR is then undertaken. 

1 . 1 U.L of each first-round reaction is diluted in 1 mL of sterilized/dionized water (1:1 000 
dilution) to make the DNA template (see Note 8). 

2. A further PCR is set up as above (Subheading 3.4.1., steps 1-4) using the nested (or 
seminested) primers and 2 uL of the diluted template made above. 

3. When the reaction is completed, run out at least 10 uL of each sample on a 1% agarose gel 
in the presence of ethidium bromide. 

4. Any band(s) of interest should be remade from the original vectorette library through the 
full-nested LDV PCR protocol, i.e., 65 cycles as before. If the same PCR product is 
observed again it should be cloned and/or directly sequenced. Suitable positive controls 
should also be employed (see Note 9). If nonspecific bands keep appearing then steps 
should be taken to eradicate these (see Note 10). 

3.5. Sequencing LDV PCR Product 

Specific bands from LDV PCR can be directly sequenced if prepared correctly. A 
protocol is described to purify and to directly sequence LDV PCR products using the 
Big Dye Terminator Cycle sequencing ready reaction kit (Applied Biosystems). For 
longer products of several kilobases in length, it may be preferable to first clone and 
then sequence the bands of interest. 

1. Run out 25 uL of the product of interest on an ethidium bromide stained 0.9% agarose 
gel. When the band of interest is viewed under UV light and is visibly separated from 
primers (and any other possible bands, such as known germline products), use a clean 
scalpel to cut out the smallest possible slice of agarose containing the band. 

2. Purify the band using the QIAquick Gel Extraction kit, microcentrifuge protocol (Qiagen) 
and elute into 30 uL of elution buffer. This gives the template for the sequencing reaction 
(see Note 11). 

3. For direct sequencing from the Vectorette end of a LDV PCR product internal Vectorette II 
sequencing primers are available (Sigma-Genosys, The Woodlands, TX). Vectorette 
sequencing primers are supplied lyophilized at 500-pmol concentration. These should 
be resuspended in sterilized/deionized water at a suitable working concentration for a cycle 
sequencing reaction, in this case 1.6 pmol/uL. 

4. It is also possible to use the nested (nIP) primer used in the second round of PCR to 
sequence from the known end of the LDV PCR product to check that the LDV PCR did 
initiate from the correct location. 

5. Set up a Big Dye sequencing reaction, depending on the type of thermal cycler being 
used, the following reaction should be made up in an appropriate tube: 1 u.L of sequenc- 
ing primer (1.6 pmol/uL), 5 uL of the LDV PCR product, and 4 uL of Terminator Ready 
Reaction Mix to give a 10-uL total volume (see Note 12). 

6. The standard automated sequencing protocol should be followed (see Note 12). 



282 Fenton, Pratt, and Morgan 

3.6. Cloning LDV PCR Products 

Cloning methods need not be described in great detail, but we have successfully 
used the pT7Blue Perfectly Blunt Cloning System (Novagen) to clone LDV PCR prod- 
ucts. Candidate LDV PCR products can be either gel purified, e.g., QIAquick Gel 
Extraction kit (Qiagen) or column purified, e.g., Wizard PCR Preps DNA purification 
system (Promega). Obviously, once cloned into a plasmid, the insert can be sequenced 
from either side with primers flanking the cloning site or using internal primers that 
bind to the known sequence of the PCR product, which includes the vectorette sequenc- 
ing primer since the vectorette unit is still ligated to the insert. 

4. Notes 

1. High-molecular-weight genomic DNA can be prepared from samples by proteinase K 
digestion, phenol-chloroform extraction, and ethanol precipitation (8). To quantify the 
genomic DNA to be used in LDV PCR, measure it on a spectrophotometer and check the 
quality via a 260/280 nm ratio reading (8). If the integrity of the DNA is doubted, then a 
standard long PCR with control primers can be attempted first to prove that large PCR 
products can be made from this template. 

2. The greater the number of vectorette libraries made per sample, the greater the chance of 
there being a restriction site (with a Vectorette unit ligated to it) within the range of which 
the polymerase enzyme can reach from the anchoring (IP) primers designed. Anything 
between 5-8 vectorette libraries is recommended depending on the amount of DNA avail- 
able. The obvious restriction enzymes to use are Hindlll, BamHl, EcoRl, Clal (for which 
compatible Vectorette units are available). For the blunt Vectorette unit, which is also 
available, we have successfully employed blunt cutter enzymes such as PvuU, Rsal, and 
Hindi. It is also possible to use enzymes which recognize the same restriction sites as the 
other enzymes, e.g., Bglll with a BamHl Vectorette unit. 

3. The DNA polymerase enzyme used will be extremely important, since it is required to 
generate as long a PCR product as possible. The further one can amplify along a PCR 
fragment from the initial first round of the PCR the more chance there is of reaching an 
appropriate restriction site with a vectorette unit ligated to it. For the PCR, we have 
succesfully used both LA Taq (Takara, Japan) and Elongase (Gibco-BRL) enzyme sys- 
tems. Other workers have reported the successful use of a Boehringer hi-fidelity enzyme 
system in vectorette PCR. 

4. The temperature cycling for the vectorette ligation step makes the process more efficient. 
Restriction enzyme binding sites are reformed when target DNA fragments ligate to each 
other but not when they ligate to the appropriate vectorette unit, therefore this tempera- 
ture cycling increases the relative proportion of target DNA correctly linked to vectorette 
units. Further digestion therefore makes more compatible DNA available to ligate to 
vectorette units. 

5. When possible, it is recommended that a two-step PCR protocol where annealing and 
denaturation are undertaken at the same temperature after a denaturation step. The T m of 
the Vectorette PCR primers are relatively high (both are given as >70°C), however, a 
separate annealing step can be employed. We have successfully utilized the more conven- 
tional thermal cycling profile of three separate temperature steps typically used in stan- 
dard PCR, with primers known to have lower annealing temperatures, e.g., 60°C or 62°C. 
In such a case, the extension step is still undertaken at 68°C, giving a typical cycling 
profile of 95°C for 3 min, followed by cycles consisting of denaturation at 95°C for 45 s, 



LDVPCR 283 

annealing at 60 or 62°C for 45 s, and extension at 68 C C for 4 min. The reaction is com- 
pleted with a hold for final extension at 72°C for 10 min and a final hold at 4°C until the 
tubes are removed. Shorter or longer extension times can be employed as desired. 

6. It is easier to make up master mix (in excess by at least one tube), which contains all the 
common component reagents of a reaction, dNTP, buffer, magnesium solution, primers 
and water and add 28 uL of this master mix to each reaction tube. 2 [iL of each DNA 
library is then added to the appropriate tube and then the mineral oil placed on top. 

7. A hot start protocol is recommended for all the PCRs. This can be undertaken in one of 
several ways. First, for a manual hot start, as described in Subheading 3.4.1., namely 
adding the polymerase predissolved in water to the rest of the reaction components. This 
method can only be followed if a mineral oil overlay is being used so that the enzyme is 
loaded through the oil layer when the tube and its contents are held at the initial 95°C. 
The 20-uL volume of this addition is used solely to minimize pipeting variations. Second, 
a variation of this protocol can be utilized, especially when employing thermal cyclers 
such as the Gene AMP PCR System 9700 (Applied Biosystems) which incorporate heated 
lids so that mineral oil is not required. All the contents of the PCR are mixed together, 
including the Taq polymerase, with the reaction tube being kept on ice. The thermal cycler 
is then programmed to have an initial hold step at 4°C (for say 30 s) before the initial, 
3 min 95°C denaturation step. The tubes are then transferred from the ice to the cycler 
when it is held at 4 C C. The profile then proceeds to heat up to 95°C for the first denatur- 
ation step, thus also giving an effective hot start protocol. 

8. We have found that a 1:1000 dilution was the optimal dilution to produce a substrate for 
the second round (nested) PCR, but this can be varied, e.g., a 1:10,000 or 1:100 dilution. 

9. When an appropriate known restriction enzyme recognition site is within range LDV 
PCR will amplify a band of predictable size from germline DNA, which can give a good 
positive control band and prove that the initiating primers designed are good and working. 
When looking at recombination events it may be advisable to perform the LDV PCR proce- 
dure with libraries made from germline DNA (e.g., placenta), as well as the sample of 
interest. Any germline bands will be observed in both lanes when the products are run out 
side by side on a gel. However, a band of different size will result from DNA that has 
undergone a recombination event. In the case of a translocation, this will provide the actual 
translocation breakpoint that will obviously not be observed with a germline template. 

10. As was discussed in Subheading 1. spurious amplification can occur. If this is observed 
then the hot start protocol used should be evaluated and if necessary revised. Spurious 
bands can very infrequently be observed in vectorette libraries made with a blunt cutting 
restriction enzyme after incorrect ligation has apparently occurred (JF personal experi- 
ence!), although this has never presented itself as a major problem. 

1 1 . The concentration of the PCR product is critical to the sequence reaction. An easy way to 
determine that there is enough LDV PCR product for sequencing is to do a quick "eye- 
ball" method check. Simply run a very small volume of the purified product, say 4 ^L, 
out on a 1 % agarose minigel and check that the correct band is clearly visible to the 
naked eye when illuminated with UV light in the presence of ethidium bromide. 

12. The reaction described is half the standard volume recommended by Applied Biosystems. 
The relatively high concentration of primer allows a greater volume to be added for the 
template. Good, clean sequence can be obtained from a direct sequencing protocol. 
However, if a bigger band (several kb in length) is required to be sequenced it may be 
necessary to clone this product into a suitable vector {see Subheading 3.5.2.) and then 
sequence it. This is because up to 100 times more of a 2000 bp product (a typical LDV 



284 Fenton, Pratt, and Morgan 

PCR product size), is required in a sequencing reaction than say a smaller 200 bp prod- 
uct. Therefore, it may be difficult to get a high enough concentration of this band in the 
sequencing reaction. 

Acknowledgment 

Vectorette is a trade mark of Sigma-Genosys. The development of the LDV PCR 
technique in our lab was supported by a grant from Yorkshire Cancer Research. 

References 

1. Riley, J., Butler, R., Ogilvie, D., Finnear, R., Jenner, D., Powell, S., et al. (1990) A novel, 
rapid method for the isolation of terminal sequences from yeast artificial chromosome 
(YAC) clones. Nucl. Acid Res. 18, 2887-2890. 

2. Arnold, C. and Hodgson, I. J. (1991) Vectorette PCR: A novel approach to genomic walk- 
ing. PCR Meth. Appl. 1, 39-42. 

3. Chenchik, A., Diachenko, L., Moqadam, F., Tarabykin, V., Lukyanov, S., and Siebert, P. 
D. (1996) Full-length cDNA cloning and determination of mRNA 5' and 3' ends by ampli- 
fication of adaptor-ligated cDNA. Biotechniques 21, 526-534. 

4. Mills, K. I., Sproul, A. M., Ogilvie, D., Elvin, P., Leibowitz, D., and Burnett, A. K. (1992) 
Amplification and sequencing of genomic breakpoints located within the M-bcr region by 
Vectorette-mediated polymerase chain reaction. Leukemia 6, 481-483. 

5. Zhang, J. G., Goldman, J. M., and Cross, N. C. P. (1995) Characterization of genomic BCR- 
ABL breakpoints in chronic myeloid leukemia by PCR. Br. J. Haematol. 90, 138-146. 

6. Proffitt, J., Fenton, J., Pratt, G., Yates, Z., and Morgan, G. (1999) Isolation and 
characterisation of recombination events involving immunoglobulin heavy chain switch 
regions in multiple myeloma using long distance vectorette PCR (LDV-PCR). Leukemia 
13, 1100-1107. 

7. Hengen, P. N. (1995) Methods and reagents — Vectorette, splinkerette and boomerang 
DNA amplification. TIBS 20, 372-373. 

8. Hui, E. K., Wang, P., andLo, S. J. (1998) Strategies for cloning unknown cellular flanking 
DNA sequences from foreign integrants. Cell. Mol. Life Sci. 54, 1403-1411. 

9. Sambrook, J., Fristsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory 
Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 
9.16-9.19. 



29 



Nonspecific, Nested Suppression PCR Method 
for Isolation of Unknown Flanking DNA 
("Cold-Start Method") 

Michael Lardelli 
1. Introduction 

The ability of the polymerase chain reaction (PCR) to amplify DNA depends upon 
the existence of defined primer binding sites. Thus, to amplify a region of flanking 
unknown DNA sequence, a defined primer binding site must be created. Numerous 
strategies have been found to do this, such as addition of nucleotides or ligation of 
oligonucleotides to DNA ends, restriction of the flanking DNA followed by ligation of 
known DNA sequences and, (for cDNA amplification), ligation of RNA oligonucle- 
otides to the RNA molecule followed by reverse transcription PCR (reviewed in 
ref. 1). All of these methods require considerable molecular biological processing of 
the source nucleic acid. 

A far simpler strategy is to use nonspecific binding by oligonucleotides to generate 
a primer binding site in unknown flanking DNA (see Fig. 1). Numerous variations on 
this strategy have been described (2-5). A general problem with these strategies is 
detection and purification of the desired product from among numerous spurious 
amplification products. However, a method has now been developed that is very simple 
to perform, can generate long PCR fragments, requires no processing of the source 
DNA other than PCR and produces few spurious products. Because of its simplicity 
and sensitivity it can be used as the method of first choice before resorting to more 
complex procedures. 

The "cold-start method" consists of two sequential PCRs. In the first PCR 
("nonspecifically primed PCR," NSPPCR), a single primer binding to known sequence 
and priming toward unknown flanking DNA is used under conditions of low specific- 
ity, e.g., low annealing temperature. The intention is to generate DNA strands primed 
from within the unknown region and extending past the primer binding site in the 
known DNA region. In the following PCR cycle, the primer can now bind to the spe- 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

285 



286 



Lardelli 



Symbol Key 



UJJLU 



Known DNA sequence 



Unknown flanking 
DNA sequence 

Non-specific priming 
by first primer 

Primer binding site 
in known DNA 




Specific priming by 

1 st primer. 

Priming by extended primer 

Terminal mismatch of 
extended primer 

Priming by truncated 
extended primer 



Reaction 1 - NSPPCR 



Reaction 2 - Reamplification PCR 



Before exonuclease truncation of extended primer: 



No priming in unknowr 
sequence due to terminal mismatch 



After partial exonuclease truncation of extended primer: 



IP 



Priming occurs when the extended 
primer has been sufficiently truncated 

Fig. 1. The "cold-start method" for cloning flanking unknown sequences. The method 
consists of two sequential PCRs separated by a dilution step. 



Cold-Start Method to Isolate Unknown DNA 287 

cific site in the known sequence and, by generating the complement of the first DNA 
strand, will produce a perfect primer binding site at the other end of the DNA molecule 
(see Fig. 1). This DNA fragment will now amplify exponentially during subsequent 
PCR cycles. Numerous spurious DNA fragments can be generated during this first 
PCR, but the desired flanking DNA fragments begin with the advantage that they 
already possess one perfect primer annealing site and so they are amplified at rela- 
tively high frequency. 

Some methods that rely on non-specific priming use a primer binding specifically 
in the known sequence region and a second primer binding nonspecifically in the 
unknown flanking region. The advantage of using a single primer for both purposes is 
that inverted repeats are generated at both termini of the PCR products and so the 
reaction becomes a suppression PCR (6). Normally, amplification by conventional 
PCR favors short fragments over longer ones. In a nonspecific PCR, this leads to a 
predominance of shorter fragments among the products. However, suppression PCR 
counters this effect because, in shorter fragments, the terminal inverted repeats anneal 
at high frequency, thus blocking primer annealing and DNA synthesis. Low primer 
concentrations can enhance this suppression effect. 

The second PCR of the Cold-start method ("reamplification PCR") specifically 
amplifies the desired product(s) from the NSPPCR. To do this, the products of the 
NSPPCR are diluted and then a novel form of seminested PCR is performed. Nor- 
mally, seminested PCR could not specifically amplify the desired products because 
the only primer known to bind in the unknown flanking region is the initial primer. 
The initial primer binds at both ends of all the products of the NSPPCR. Any subse- 
quent PCR using this primer amplifies both desired and spurious products. However, 
we discovered that by extending the initial primer by six nucleotides and using a proof- 
reading thermostable DNA polymerase, amplification of the desired products is greatly 
favored. Presumably, during the reamplification PCR, the proofreading polymerase 
amplifies the desired fragment in a linear manner until the primer is truncated suffi- 
ciently by the polymerase to bind at both ends of the desired fragment after which 
exponential amplification occurs (see Fig. 1). Meanwhile, partial binding of the 
extended primer at the ends of spurious products inhibits their reamplification. 
Reamplification PCR is conducted under stringent conditions and is also a form of 
suppression PCR so that it favors the amplification of longer fragments. 

In its simplest form, the cold-start method consists of two PCRs separated by a 
dilution step. The number of final products is small and the desired products can be 
identified by Southern blotting against the known DNA sequence or simply by cloning 
and sequencing. There are a number of optional enhancements of the method that can 
further increase its success rate. The basic method is described here and references are 
given for the enhancements. 

2. Materials 

1. Oligonucleotide primers for NSPPCR and reamplification PCR (see Subheading 3.1. 
Primer Design below). 

2. A DNA solution including the known DNA region with desired flanking sequences. 

3. Double-distilled water. 



288 Lardelli 

4. Thermostable DNA polymerase lacking 3'-5' exonuclease activity and recommended 10X 
concentrated reaction buffer (e.g., Taq DNA polymerase and associated buffer from 
Stratagene Cloning Systems, La Jolla, CA). 

5. Thermostable DNA polymerase possessing 3'-5' exonuclease activity and recommended 
10X concentrated reaction buffer (e.g., Pfu Turbo® DNA polymerase from Stratagene 
Cloning Systems). 

6. 10 mM dNTPs (2.5 mM each of dATP , dCTP, dGTP, and dTTP). 

7. Paraffin oil (if performing reactions under oil, see Note 1). 

8. PCR thermal cycler and accessories (e.g., a thermal cycler that accepts 96-well microassay 
plates with paraffin oil such as the PTC-200 Peltier Thermal Cycler with Multiplate™ 96 
Polypropylene V-Bottom Microplates from MJ Research Inc., Watertown, MA). 

9. Micropipeters and pipet tips (e.g., Gilson, France) for liquid. 

10. 1.5-mL capped, polypropylene microfuge tubes. 

11. 6X gel loading buffer: 15% w/v Ficoll (Type 400; Amersham Pharmacia Biotech AB, 
Uppsala, Sweden), 0.35% w/v Orange G (Sigma, St. Louis, MO), 60 mM ethylene dia- 
minetetraacetic acid (EDTA), pH 8.0. 

12. Equipment and reagents for agarose gel electrophoresis. 

13. DNA electrophoresis size markers (e.g., 1 Kb DNA Ladder; Life Technologies, Rockville, MD). 

14. Reagents for Southern blotting, radioactive probe synthesis and hybridization, and 
posthybridization washing. 

15. A scalpel for excision of DNA-containing bands from agarose gels. 

16. Reagents for purification of DNA from agarose gel (e.g., QIAquick PCR Purification Kit, 
QIAGEN GmbH, Hilden, Germany). 

17. Reagents for cloning of blunt-ended DNA fragments (e.g., the Zero Blunt™ TOPO® PCR 
Cloning Kit from Invitrogen Corporation, Carlsbad, CA). 

3. Methods 

3. 1. Primer Design 

Two primers are required for this procedure, "first primer" and "extended primer". 
The first primer should have a melting temperature (r m ) of around 60°C and be around 
20 nucleotides in length. Ideally, it should be designed to bind 100-200 bp from the 
unknown flanking DNA and it must prime towards this DNA. The extended primer is 
identical to the first primer but is extended at its 3' end by six nucleotides correspond- 
ing to the known sequence (see Fig. 1 and Note 2). 

3.2. Nonspecifically Primed PCR 

Details are given below for one reaction. Scale up the premixes described if mul- 
tiple reactions are to be performed, e.g. to test different annealing temperatures, primer 
concentrations or Mg 2+ concentrations for NSPPCR (see Note 3). As a guide to when 
NSPPCR is occurring, lower the annealing temperature or raise the Mg 2+ concentra- 
tion until bands of DNA can be observed under UV light on an agarose gel stained 
with ethidium bromide. 

1. Assemble premixes as follows: a) PCR premix: 8 uL of water, 2 uL of the DNA source 
(e.g., 20 ng of genomic DNA, see Note 4), 2 u.L of 10X Taq DNA polymerase buffer 
(e.g., 500 mM KC1, 100 mM Tris-HCl pH 8.3), 5 uL of 2.5-20 mM MgCl 2 (see Note 3), 
1 uL of first primer @ 2.5-40 \xM (see Note 5), 2 uL dNTPs (10 mM). b) Polymerase 



Cold-Start Method to Isolate Unknown DNA 289 

premix: 0.5 [xL of 10X Taq DNA polymerase buffer, 4. 1 u,L of water, 0.4 (xL of Taq DNA 
polymerase (5 U/u.L) (see Note 6). 

2. Place the premix in a well of a PCR microassay plate or in a PCR tube. Cover with one 
drop of oil (see Note 7). 

3. Place the plate or tube in the PCR thermal cycler and heat to 94°C (e.g., "pause" the 
cycler during the first denaturation step of the PCR cycling protocol given in step 4 
below). Wait 30 s and then eject the polymerase premix into the plate well / tube from just 
above the oil (e.g., onto the wall of the plate well or tube). The polymerase mix will fall 
through the oil and mix with the other PCR components (see Note 1). 

4. Perform PCR cycling as follows: 35 cycles: Denaturation at 94°C for 30 s, annealing at 
5 to 30°C below the oligonucleotide's calculated annealing temperature for 1 min (see 
Note 8), temperature ramp of 0.5°C/s to 72°C then 72°C for 3 min. 

3.3. Reamplification PCR 

From the NSPPCR, it is possible to proceed directly to reamplification PCR or, if 
multiple annealing temperatures and/or Mg 2+ concentrations are being tested, one can 
examine these first for the presence of desired sequences by Southern hybridization 
(see Note 3). Even if one cannot detect desired sequences by this method (because 
there is too little known sequence in the desired products against which to probe or 
because the concentration of desired products is too low) it is worthwhile to proceed 
with a reamplification PCR because this may, nevertheless, reveal desired sequences. 

1. Dilute a sample of the NSPPCR products 1 : 1000 in water. 

2. Assemble premixes as follows: a) PCR premix: 14u.L of water, 1 uToffhe 1:1000 diluted 
NSPPCR products, 2 U.L of 10X Pfu Turbo 9 DNA polymerase buffer (200 mM Tris-HCl 
pH 8.8, 20 mM MgS0 4 , 100 mM KC1, 100 mM [NH 4 ] 2 S0 4 , 1% Triton X-100, 0.1% 
nuclease-free BSA), 1 u.L of extended primer@ 10 \iM (see Note 9), 2 u,L dNTPs (10 mM). 
b) Polymerase premix: 4.3 uL of water, 0.5 |xL of 10X Pfu Turbo® DNA polymerase 
buffer, 0.2 ixL of Pfu Turbo DNA polymerase (2.5 U/uL) (see Note 10). 

3. Place the premix in a well of a PCR microassay plate or in a PCR tube. Cover with one 
drop of oil (see Note 7). 

4. Place the plate or tube in the PCR cycler and heat to 95 C C. (e.g., "pause" the PCR cycler 
during the first denaturation step of the PCR cycling protocol given in step 5 below). 
Wait 30 s, and then eject the polymerase premix into the plate well / tube from just above 
the oil (e.g., onto the wall of the plate well or tube). The polymerase mix will fall through 
the oil and mix with the other PCR components (see Note 1). 

5. Perform PCR cycling as follows: 35 cycles: Denaturation at 95°C for 30 s, annealing at 
5 to 10°C below the oligonucleotide's calculated annealing temperature for 1 min, tem- 
perature ramp of 0.5°C/s to 72°C then 72°C for 4 min. 

3.4. Identification of Desired Products from the Reamplification PCR 

1. Remove a 10-u.L sample from the reamplification PCR, add 2.5 u.L of 5X loading buffer 
and conduct electrophoresis on a 1.5 % agarose gel beside size markers (e.g., 1 kb DNA 
ladder). If one or more bands are seen there are two alternative ways to proceed: 

2. (optional) The gel can be Southern blotted. Any bands containing the desired sequences 
can be detected by probing the blot using DNA corresponding to the area of known 
sequence between the primer binding site and the unknown flanking region (see ref. 7 for 
methods). When a band containing the desired sequences is identified, the remaining 
reaction products can be processed as in step 3 below (see Note 11). 



290 Lardelli 

3. The bands on the gel are excised with a scalpel and the DNA they contain is purified (e.g., 
using the QIAquick PCR Purification Kit from Qiagen GmbH), cloned using a system 
allowing cloning of blunt-ended PCR fragments (e.g., the Zero Blunt™ TOPO® PCR 
Cloning Kit from Invitrogen Corporation) and then sequenced. Desired fragments can be 
identified since they contain the area of known sequence between the primer binding site 
and the unknown flanking region. 

4. Notes 

1 . The method for "hot-starting" PCR that is described in this protocol is the optimal one for 
starting a PCR that is being conducted under paraffin oil. If you wish to hot-start the 
reaction using thermostable DNA polymerase to which a blocking antibody is initially 
bound, then the entire reaction can be made up as one solution (rather than being divided 
into reaction and enzyme premixes). 

2. The extended primer may be shortened by a number of bases at the 5' end to reduce the 
melting temperature. However, note that the primer is still required to bind at the chosen 
annealing temperature even when the additional six nucleotides at its 3' end are removed 
by the exonuclease activity of the polymerase. 

3. Multiple NSPPCRs can be performed using different annealing temperatures and Mg 2+ 
concentrations to find those conditions that give sufficient nonspecific binding by the 
first primer in the unknown flanking DNA to amplify the desired sequence. The NSPPCRs 
are then electrophoresed on an agarose gel, Southern blotted and hybridized with a probe 
containing the known sequence between the known first primer binding site and the flank- 
ing, unknown DNA (see ref. 7 for methods). When desired DNA fragments are identified 
in a particular NSPPCR, this reaction can then be used for reamplification PCR. This 
procedure has the advantage that different conditions produce NSPPCR products of dif- 
ferent sizes and the NSPPCR producing the largest fragment of flanking DNA can be 
selected (see also Note 5). However, note that reamplification PCR can amplify NSPPCR 
products that are present at levels below the level of detection of Southern hybridization. 

4. The efficiency of amplification of flanking cDNA sequences can be boosted by initial puri- 
fication of the desired cDNA on magnetic beads before NSPPCR. See ref. 8 for details. 

5. Varying the concentration of the primer can cause variation in the length of the desired 
NSPPCR products. Lower primer concentrations will tend to select for longer desired 
fragment lengths. 

6. This protocol should give final reagent concentrations of: 20 ng of genomic DNA (or 
other DNA source), 50 mM KC1, 10 mM Tris-HCl pH 8.3, 0.5-4 mM MgCl 2 , 0.1-1.6 \kM 
first primer, 0.4 mM dNTPs, and 2U of Taq DNA polymerase. 

7. It may not be necessary to use oil and/or "pausing of the PCR cycling" if the containment 
of the PCR and/or the method of "hot starting" obviate this. See also Note 1. 

8. If the annealing temperature initially used for NSPPCR does not result in amplification of 
desired products after the reamplification PCR, then lower the annealing temperature 
further. See also Note 3. 

9. Despite that the reamplification PCR is a form of suppression PCR, lowering the extended 
primer concentration has not been shown to have a great effect on the size of the 
reamplified products. Lower primer concentrations apparently simply produce less DNA. 

10. This protocol should give final reagent concentrations (not including template DNA) of: 
20 mM Tris-HCl pH 8.8, 2 mM MgS0 4 , lOmMKCl, 10 mM [NH 4 ] 2 S0 4 , 0.1% TritonX- 
100, 0.01% nuclease-free BSA, 0.4 uM extended primer, 0.4 mM dNTPs, 0.5 U Pfu Turbo 
DNA polymerase. 



Cold-Start Method to Isolate Unknown DNA 291 

11. If the probe sequence includes the primer binding site, then a low level of hybridization 
of the probe with all PCR products is possible. 

References 

1. Hui, E. K., Wang, P. C, and Lo, S. J. (1998) Strategies for cloning unknown flanking 
DNA from sequences flanking foreign integrants. Cell. Mol. Life Sci. 54, 1403-1411. 

2. Malo, M. S., Srivastava, K., Andresen, J. M., Chen, X.-N., Korenberg, J. R., and Ingram, 
V. M. (1994) Targeted gene walking by low stringency polymerase chain reaction: Assign- 
ment of a putative human brain sodium channel gene (SCN3A) to chromosome 2q24-31. 
Proc. Natl. Acad. Sci. USA 91, 2975-2979. 

3. Parker, J. D., Rabinovitch, P. S., and Burmer, G. C. (1991) Targeted gene walking poly- 
merase chain reaction. Nucleic Acids Res. 19, 3055-3060. 

4. Parks, C. L., Chang, L.-S., and Shenk, T. (1991) A polymerase chain reaction mediated by 
a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein 
coding region. Nucl. Acids Res. 19, 7155-7160. 

5. Trueba, G. A. and Johnson, R. C. (1996) Random primed gene walking PCR: a simple 
procedure to retrieve nucleotide fragments adjacent to known DNA sequences. BioTech- 
niques 21, 20. 

6. Lukyanov, K. A., Launer, G A., Tarabykin, V. S., Zaraisky, A. G., and Lukyanov, S. A. 
(1995) Inverted terminal repeats permit the average length of amplified DNA fragments to 
be regulated during preparation of cDNA libraries by polymerase chain reaction. Analyt. 
Biochem. 229, 198-202. 

7. Sambrook, J. and Russel, D. Molecular Cloning: A Laboratory Manual, 3rd edition, Cold 
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. 

8. Tamme, R., Camp, E., Kortschak, R. D., and Lardelli, M. (2000) Non-specific, nested, 
suppression PCR method for isolation of unknown flanking DNA. BioTechniques 28, 
895-902. 



30 

Inverse PCR 

cDNA Cloning 
Sheng-He Huang 

1. Introduction 

Since the first report on cDNA cloning in 1972 (7), this technology has been devel- 
oped into a powerful and universal tool in isolation, characterization, and analysis of 
both eukaryotic and prokaryotic genes. But the conventional methods of cDNA clon- 
ing require much effort to generate a library that is packaged in phage or plasmid and 
then survey a large number of recombinant phages or plasmids. There are three major 
limitations in those methods. First, substantial amount (at least 1 pig) of purified mRNA 
is needed as starting material to generate libraries of sufficient diversity (2). Second, 
the intrinsic difficulty of multiple sequential enzymatic reactions required for cDNA 
cloning often leads to low yields and truncated clones (3). Finally, screening of a 
library with hybridization technique is time-consuming. 

Polymerase chain reaction (PCR) technology can simplify and improve cDNA clon- 
ing. Using PCR with two gene-specific primers, apiece of known sequence cDNA can 
be specifically and efficiently amplified and isolated from very small numbers (<10 4 ) 
of cells (4). However, it is often difficult to isolate full-length cDNA copies of mRNA 
on the basis of very limited sequence information. The unknown sequence flanking a 
small stretch of the known sequence of DNA cannot be amplified by the conventional 
PCR. Recently, anchored-PCR (5-7) and inverse PCR (8-10) have been developed to 
resolve this problem. Anchored-PCR techniques have the common point: DNA clon- 
ing goes from a small stretch of known DNA sequence to the flanking unknown 
sequence region with the aid of a gene-specific primer at one end and a universal 
primer at other end. Because of only one gene specific primer in the anchored-PCR it 
is easier to get a high level of nonspecific amplification by PCR than with two gene- 
specific primers (10,11). The major advantage of inverse PCR (IPCR) is to amplify 
the flanking unknown sequence by using two gene-specific primers. 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

293 



294 Huang 

■ r mllNA 

I Hl-»w^l'«.iv»r- 

r* - ' wt*t* 




Fig. 1. Diagram of IPCR for cDNA cloning. The procedure consists of five steps: reverse 
transcription, synthesis of second strand cDNA, circularization of double-strand cDNA, reopen 
the circle DNA, and amplification of reverse DNA fragment. The black and open bars represent 
the known and unknown sequence regions of double-stranded cDNA, respectively. 



At first, IPCR was successfully used in the amplification of genomic DNA seg- 
ments that lie outside the boundaries of known sequence (8,9). I have a new procedure 
which extends this technique to the cloning of unknown cDNA sequence from total 
RNA (10). Double-stranded cDNA is synthesized from RNA and ligated end to end 
(see Fig. 1). Circularized cDNA is nicked by selected restriction enzyme or denatured 
by NaOH treatment (12,13). The reopened or denatured circular cDNA is then ampli- 
fied by two gene-specific primers. Recently, this technique has been efficiently used 
in cloning full-length cDNAs (14-16). The following protocol was used to amplify 
cDNA ends for the human stress-related protein ERp72 (10) {see Fig. 2). 

2. Materials 

2.1. First-Strand cDNA Synthesis 

1. Total RNA prepared from human CCRF/CEM leukemic lymphoblast cells (17,18). 

2. dNTP mix: 10 mM of each dNTP. 

3. Random primers (Boehringer Mannheim, Indianapolis, IN). Prepare in sterile water at 
1 u.g/uL. Store at -20°C. 

4. RNasin (Promega, Madison, WI). 

5. Actinomycin D (1 mg/mL). Actinomycin D is light sensitive and toxic. It should be stored 
in a foil-wrapped tube at -20 C C. 

6. MMLV reverse transcriptase. 

7. 5X First-strand buffer: 0.25 M Tris-HCl (pH 8.3), 0.375 M KC1, 50 mM MgCl 2 , 50 mM 
dithiothreitol (DTT), and 2.5 mM spermidine. The solution is stable at -20°C for more 
than 6 mo. 



IPCR: cDNA Cloning 295 




Fig. 2. Application of IPCR to amplifying the joining region (280 bp) from 5' (160 bp) and 
3' (120 bp) sequences of human ERp72 cDNA . Amplified DNAs from CCRF/CEM cells sen- 
sitive (lane 1) and resistant (lane 2) to cytosine arabinoside stained by ethidium bromide (A) or 
hybridized with 32 P-labeled ERp72 cDNA (B). See text for the sequences of the primers and the 
parameters of IPCR. 



2.2. Second-Strand Synthesis 

1. 10X second strand buffer: 400 raM Tris-HCl (pH 7.6), 750 mM KC1, 30 mM MgCl 2 , 
100 mM (NH 4 ) 2 S0 4 , 30 mM DTT, and 0.5 mg/mL of bovine serum albumen (BSA). The 
solution is stable at -20°C for at least 6 mo. 

2. 1 mM nicotinamide adenine dinucleotide (NAD). 

3. RNase H (2 U/uL). 

4. Escherichia coli (E. coli) DNA polymerase I (5 U/(xL). 

5. E. coli DNA ligase (1 U/ixL). 

6. Nuclease-free H 2 0. 

7. T4 DNA polymerase. 

8. 200 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0. 

9. GeneClean (Bio 101 Inc., La Jolla, CA). 

10. TE buffer: 10 mM Tris-HCl, pH 7.6, 1 mM EDTA. 

11. DNA standards. Prepare 1-mL aliquots of a purified DNA sample at 1, 2.5, 5, 10, and 
20 [ig/mL in TE buffer. Store at -20°C for up to 6 mo. 

12. TE/ethidium bromide: 2 (xg/mL of ethidium bromide in TE buffer. Store at 4°C for up to 
6 mo in a dark container. 



296 Huang 

2.3. Circularization and Cleavage or Denaturation 



5X ligation buffer (supplied with T4 DNA ligase). 

T4 DNA ligase (1 U/nL). 

T4 RNA ligase (4 fig/ixL). 

15 \xM Hexaminecobalt chloride. 

Phenol:CHCl 3 :isoamyl alcohol (25:24:1). 

3 M sodium acetate, pH 7.0. 

Absolute ethanol. 

70% ethanol. 



2.4. Inverse PCR 

1. 10X PCR buffer: 100 mM Tris-HCl, pH 8.3, 500 mM KC1, 15 mM MgCl 2 , 0.01%(w/v) 
gelatin. 

2. 15mMMgCl 2 . 

3. Deoxyoligonucleotides were synthesized on an Applied Biosystems (Foster City, CA) 
380B DNA synthesizer and purified by OPEC column from the same company. The 
primer pairs were selected from the 5' and 3' sequence of the cDNA coding for human 
ERp72 stress-related protein (5'-primer: 5'-TTCCTCCTCCTCCTCCTCTT-3'; 3'-primer: 
5'- ATCTAAATGTCTAGT-3') (10). 

4. Light mineral oil. 

5. Taq DNA polymerase. 

3. Methods 

3.1. First-Strand cDNA Synthesis (19) 

1. Perform reverse transcription in a 25-u.L reaction mixture, adding the following com- 
ponents: 

5X first-strand buffer 5.0 fxL 

dNTP mix 2.5 \xL 

random primers 2.5 (xL 

Rnasin 1.0 U 

actinomycin D 1.25 u.L 

MMLV reverse transcriptase 250 U 
RNA 15-25 fig of total RNA (Heat denature RNA at 65°C for 

3 min prior to adding to reaction) 
Nuclease-free H 2 to 25 u.L final vol. 

3.2. Second-Strand Synthesis (20) 

1. Add components to the first-strand tube on ice in the following order: 
10X second-strand buffer 12.5 (J.L 

ImMNAD 12.5 uL 

RNase H (2 ii/uL) 0.5 uL 

E. coli DNA polymerase I ( 5 (/uL ) 5.75 uL 
E. coli ligase (lfx/jiL) 1.25 (xL 

nuclease-free water 92.5 |xL 

2. Incubation at 14°C for 2 h. 

3. Heat the reaction mix to 70°C for 10 min, spin for few seconds, and then put in ice. 

4. Add 4 U of T4 DNA polymerase and incubate at 37°C for 10 min to blunt the ends of 
double-stranded cDNA. 



IPCR: cDNA Cloning 297 

5. Stop the reaction with 12.5 uL of 0.2 M EDTA and 200 u.L sterile H 2 0. 

6. Concentrate and purify the sample with Geneclean. Resuspend the DNA in 100-200 (xL 
of sterile H 2 0. 

7. Estimate the DNA concentration by comparing the ethidium bromide fluorescent inten- 
sity of the sample with that of a series of DNA standards on a sheet of plastic wrap (21). 
Dot 1-5 |xL of sample onto plastic wrap on a UV transilluminator. Also dot with 5 (xL of 
DNA standards. Add an equal volume of TE buffer containing 2 u,g/mL of ethidium bro- 
mide, mix by repipeting up and down. Use proper UV shielding for exposed skin and eyes. 

3.3. Circularization and Cleavage (see Notes 1-4) 

1. Set up the circularization reaction mixture (150 u,L) containing the following components: 
100 [xL (100 ng DNA) of the purified sample, 30 uL of 5X ligation buffer, and 6 [xL of T4 
DNA ligase. Finally, add 2 (xL of T4 RNA ligase or 15 u,L of 15 \iM hexaminecobalt 
chloride (see Note 5). 

2. Incubate at 18°C for 16 h. 

3. Boil the ligated circular DNA for 2-3 min in distilled water or digest with an appropriate 
restriction enzyme to re-open circularized DNA. 

4. Purify the DNA sample with Geneclean as described in step 6 in Subheading 3.2. or 
extract with water-saturated phenol/CHCl 3 and then precipitate with ethanol (20). 

3.4. Inverse PCR (see Note 6) 

1 . Add 1/10 of the purified cDNA to 100 u.L of amplification mixture (22): 
10X PCR buffer 10 [xL 

15mMMgCl 2 10 [xL 

dNTP mix (2.5 mM of each) 10 [xL 

5'-primer (10 pmole/u,L) 10 u,L 

3'-primer (10 pmole/u,L) 10 u,L 

cDNA 10 uL 

Nuclease-free H 2 39.5 [xL 

Taq DNA polymerase (2.5 (x/jxL) 0.5 [xL 

2. Cap and vortex the tubes to mix. Spin briefly in a microfuge. Cover each reaction with a 
few drops of light mineral oil to prevent evaporation. 

3. Put a drop of mineral oil into each well of the thermal cycler block that will hold a tube. 
Load the reaction tubes. 

4. Amplify by PCR using the following cycle profile: 
25 cycles 94°C 1 min (denaturation) 

65°C 2 min (annealing) 
72°C 4 min (elongation) 

4. Notes 

1. For maximum efficiency of intra-molecular ligation, low concentration of cDNA should 
be used in the ligation mix. High density of cDNA may enhance the level of heteroge- 
neous ligation, which creates nonspecific amplification. 

2. Cleavage or denaturation of circularized double-strand cDNA is important because circu- 
lar double-strand DNA tends to form supercoil and is poor template for PCR (23). Circu- 
larized double-strand DNA is only good for amplification of a short DNA fragment. 

3. The following three ways can be considered to introduce nicks in circularized DNA. Boil- 
ing is a simple and common way. Owing to the unusual secondary structure of some 



298 Huang 

circular double-strand DNA, sometimes this method is not sufficient in nicking and dena- 
turing circular double-strand DNA. A second method is selected restriction enzyme diges- 
tion. The ideal restriction site is located in the known sequence region of cDNA. In most 
cases, it is difficult to make the right choice of a restriction enzyme because the restric- 
tion pattern in unidentified region of cDNA is unknown. If an appropriate enzyme is not 
available, EDTA-oligonucleotide-directed specific cleavage may be tried (24,25). Oligo- 
nucleotide linked to EDTA-Fe at T can bind specifically to double-stranded DNA by 
triple-helix formation and produce double-stranded cleavage at the binding site. 

4. Alkali denaturation has been successfully used to prepare plasmid DNA templates for 
PCR and DNA sequencing (12,13,26). This method should be feasible in denaturing cir- 
cularized double-strand cDNA. 

5. Inclusion of T4 RNA ligase or hexaminecobalt chloride can enhance the efficiency of 
blunt-end ligation of double-strand DNA catalyzed by T4 DNA ligase (27). 

6. IPCR can be used to efficiently and rapidly amplify regions of unknown sequence flank- 
ing any identified segment of cDNA or genomic DNA. This technique does not need 
construction and screening of DNA libraries to obtain additional unidentified DNA 
sequence information. Some recombinant phage or plasmid may be unstable in bacteria 
and amplified libraries tend to lose them (23). IPCR eliminates this problem. 

Acknowledgments 

The author would like to acknowledge Dr. J. Holcenberg for his invaluable com- 
ments and generous support. The author especially thanks C.-H. Wu and B. Cai for 
their technical assistance. 

References 

1. Verma, I. M., Temple, G. F., Fan, H., and Baltimore, D. (1972) In vitro synthesis of double- 
stranded DNA complimentary to rabbit reticulocyte 10S RNA. Nature 235, 163-169. 

2. Akowitz, A. and Mamuelidis, L. (1989) A novel cDNA/PCR strategy for efficient cloning 
of small amounts of undefined RNA. Gene 81, 295-306. 

3. Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T., and Arai, K. (1987) 
High-efficiency cloning of full-length cDNA; Construction and screening of cDNA expres- 
sion libraries for mammalian cells. Meth. Enzymol. 154, 3-28. 

4. Brenner, C. A., Tarn, A. W., Nelson, P. A., Engleman, E. G., Suzuki, N., Fry, K. E., and 
Larrick, J. W. (1989) Message amplification Phenotyping ( MAPPing ): a technique to 
simultaneously measure multiple mRNAs from small numbers of cells. BioTechniques 
7, 1096-1103. 

5. Frohman, M. A. (1990) RACE: Rapid amplification of cDNA ends, in: PCR Protocols: A 
Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, 
T. J., eds.). Academic, San Diego, CA, pp 28-38. 

6. Shyamala, V. and Ames, G. F.-L. (1989) Genome walking by single-specific-primer 
polymerase chain reaction : SSP-PCR. Gene 84, 1-8. 

7. Huang, S.-H., Jong, A. Y., Yang, W., and Holcenberg, J. (1993) Amplification of gene 
ends from gene libraries by PCR with single-sided specificity. Meth. Mol. Biol. 15, 
357-363. 

8. Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse 
polymerase chain reaction. Genetics 120, 621-625. 



IPCR: cDNA Cloning 299 

9. Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) A procedure for in vitro amplification of 
DNA segments that lie outside the boundaries of known sequences. Nucl. Acids Res. 16, 8186. 

10. Huang, S.-H., Hu, Y. Y., Wu, C.-H. and Holcenberg, J. (1990) A simple method for direct 
cloning cDNA sequence that flanks a region of known sequence from total RNA by apply- 
ing the inverse polymerase chain reaction. Nucl, Acids Res. 18, 1922. 

11. Delort, J., Dumas, J. B., Darmon, M. C, and Mallet, J. (1989) An efficient strategy for 
cloning 5' extremities of rare transcrips permits isolation of multiple 5'-untranslated regions 
of rat tryptophan hydroxylase mRNA. Nucl. Acids Res. 17, 6439-6448. 

12. Cusi, M. G., Cioe', L., and Rovera, G. (1992) PCR amplification of GC-rich templates 
containing palindromic sequences using initial alkali denaturation. BioTechniques 12, 
502-504. 

13. Lau, E. C, Li, Z.-Q., and Slavkin, S. C. (1993) Preparation of denatured plasmid tem- 
plates for PCR amplification. BioTechniques 14, 378. 

14. Green, I. R. and Sargan, D. R. (1991) Sequence of the cDNA encoding ovine tumor necro- 
sis factor-a: problems with cloning by inverse PCR. Gene 109, 203-210. 

15. Zilberberg, N. and Gurevitz, M. (1993) Rapid Isolation of full length cDNA clones by 
"Inverse PCR:" purification of a scorpion cDNA family encoding a-neurotoxins. Analyt. 
Biochem. 209, 203-205. 

16. Austin, C. A., Sng, J.-H., Patel, S., and Fisher, L. M. (1993) Novel HeLa topoisomerase II 
is the lip isoform: complete coding sequence and homology with other type II 
topoisomerases. Biochim. Biophys. Acta 1172, 283-291. 

17. Delidow, B. C, Lynch, J. P., Peluso, J. J., and White, B. A.(1993) Polymerase Chain 
Reaction: Basic Protocols. Meth. Mol. Biol. 15, 1-29. 

18. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic Methods in Molecular Biology, 
Elsevier Science, New York. 

19. Km, M. S. and Berger, S. L. (1987) First strand cDNA synthesis primed by oligo(dT). 
Meth. Enzymol. 152, 316-325. 

20. Promega (1996) Protocols and Applications 3rd ed., pp. 179-190. 

21. Sambrook, J., Fritch, E. F., and Maniatis, T. (1989) Molecular Cloning, 2nd ed., Cold 
Spring Harbor Laboratory Press, New York. 

22. Saiki, R. K., Gelfand, D. H., Stoffel, S„ Scharf, S. J., Higuchi, R., Horn, G. T„ Mullis, K. 
B., and Erlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a ther- 
mostable DNA polymerase. Science 239, 487-491. 

23. Moon, I. S. and Krause, M. O. (1991) Common RNA polymerase I, II, and III upstream 
elements in mouse 7SK gene locus revealed by the inverse polymerase chain reaction. 
DNA Cell Biol. 10, 23-32. 

24. Strobel, S. A. and Dervan, P. B. (1990) Site-specific cleavage of a yeast chromosome by 
oligonucleotide-directed triple-helix formation. Science 249, 73-75. 

25. Dreyer, G. B. and Dervan, P. B. (1985) Sequence-specific cleavage of single-stranded 
DNA: Oligodeoxynucleotide-EDTA.Fe(II). Proc. Natl. Acad. Sci. USA 82, 968-972. 

26. Zhang, H., Scholl, R., Browse, J., and Somerville, C. (1988) Double strand DNA sequenc- 
ing as a choice for DNA sequencing. Nucl. Acids Res. 16, 1220. 

27. Sugino, A., Goodman, H. M., Heynecker, H. L., Shine, J., Boyer, H. W., and Cozzarelli, 
N. R. (1977) Interaction of bacteriophage T4 RNA and DNA ligases in joining of duplex 
DNA at base-paired ends. ./. Biol. Chem. 252, 3987. 



31 

Inverse PCR 

Genomic DNA Cloning 

Ambrose Y. Jong, Anna T'ang, De-Pei Liu, and Sheng-He Huang 

1. Introduction 

Inverse PCR (IPCR) was designed for amplifying anonymous flanking genomic 
DNA regions (1,2). The technique involves the digestion of source DNA, circulation 
of restriction fragments, and amplification using oligonucleotides that prime the DNA 
synthesis directed away from the core region of a known sequence, i.e., opposite of the 
direction of primers used in normal or standard PCR (Fig. 1). Prior to the invention of 
the polymerase chain reaction (PCR), the acquisition of a specific DNA fragment usu- 
ally entailed the construction and screening of DNA libraries, and the traditional 
"walking" into flanking DNA fragments involved the successive probing of libraries 
with clones obtained in the prior screening. These time-consuming procedures could 
be replaced by IPCR. Because IPCR can be used to efficiently and rapidly amplify 
regions of unknown sequence flanking any identified segment of cDNA or genomic 
DNA, researchers do not need to construct and screen DNA libraries to obtain 
additional unidentified DNA sequence information using this technique. Some 
recombinant phage or plasmids may be unstable in bacteria and amplified libraries 
tend to lose them. IPCR eliminates this problem. 

The IPCR is an effective method for identifying flanking cDNA and genomic DNA 
segments that lie outside the primers without any information (1-3). Therefore, it has 
been widely used for the identification of flanking DNA sequences from simple or 
complex genomes (4), or for obtaining promoter sequences (5,6). For example, IPCR 
can be used for the detection of adjacent DNA fragments in bacterial chromosomes 
(7), or for amplifying Tn transposon insertions (8). Several modified applications of 
IPCR have been created. For example, inverse PCR mutagenesis can generate epitope- 
tagged proteins (9), create an internal deletion (10), make a deletion library (//), or 
construct a regional peptide library (12). 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

30 1 



302 Jong et al. 

IPCR 

DNA 
5' unknown 3' unknown 



1 



RE ± RE Digestion RE 



X Self-Ligation 
+ 5' GSP 




I 



PCR 



Fig. 1. Diagram of IPCR for genomic DNA cloning. The procedure consists of four steps: 
genomic DNA isolation, circularization of double-stranded DNA, reopening of the circular 
DNA, and amplification of reverse DNA fragment. The black and open bars represent the known 
and unknown sequence regions of double-stranded cDNA, respectively. RE: restriction 
enzymesite; 65p: gene-specific primer. 



IPCR is relatively simple because intramolecular ligation can be achieved at low 
DNA concentration. One feature of IPCR is the involvement of ligation of separated 
regions at the ends of a sequence (a restriction fragment or a PCR product). This tech- 
nique can be applied to closely linked polymorphisms. Haplotypes consisting of het- 
erozygous polymorphisms have been deteremined by pedigree analysis. However, 
using inverse PCR, the linked polymorphisms can be analyzed by a single procedure 
using allele-specific primers (13). A modified protocol, long inverse PCR (LI-PCR), 
has been used to generate up to 10 kb flanking DNA segments (14). Another modified 
protocol, partial inverse PCR (PI-PCR), has been performed using a Sau3Al or other 
4-base cutter enzymes to do partial digestion of genomic DNA before the religation 
step (15). 

Recent genomic projects have made significant progress on the generation of physi- 
cal markers for the refinement of genetic maps, determination of the linear sequences 
of chromosomes, and identification of short portions of cDNA clones from mRNA 
(expressed sequence tags, or ESTs) from eukaryotic to prokaryotic organisms. PCR 
has been a useful tool for amplifying specific single-copy DNA sequences from total 
genomic DNA. In the post-genomic era, to find appropriate methods for sequencing 
the two DNA fragments adjacent to an already sequenced gene or DNA fragment in 



IPCR: Genomic DNA Cloning 303 

12 3 4 




Fig. 2. Electrophoresis of the amplified products of the IPCR on 2% agarose gel. Lane 1: 
DNA marker, 100-bp DNA ladder, the most intense band is 500 bp. Lanes 2 and 3: the DNA 
from human KB-466 cells was digested by Taql, ligated, and used as IPCR template. Lane 4: 
the KB-466 DNA was digested by Sspl, ligated, and used as IPCR template. 



eukaryotic or prokaryotic genomes is of significant importance. Needless to say, IPCR 
becomes useful in identifying flanking regions of a known DNA fragments (Fig. 2). 
Even if the complete sequence of a particular genome is known, IPCR is still a power- 
ful tool; for example, IPCR can be used to analyze the viral DNA integration sites, 
such as hepatitis B virus DNA integrating into human hepatoma cell line (16). 

In this chapter, we present a general protocol to describe how to use IPCR method 
for selectively amplifying specific genomic DNA fragments without resorting to con- 
ventional cloning procedures. 

2. Materials 

2. 1. Genomic DNA Isolation 

1. Cell lines or tissues. 

2. Premix I: 1 mL 2 M Tris-HCl, pH 7.4, 1 mL 0.5 M ethylenediaminetetraacetic acid 
(EDTA), pH 8.0, 1 mL 20% sodium dodecyl sulfate (SDS), 7 mL phosphate buffered 
saline (PBS). Prepare freshly. 

3. 10 mg/niL Proteinase K. 



304 Jong et al. 

4. Phenol/cholorform/isoamyl alcohol 25:24:1. 

5. N 250 T 10 E,: 250 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA. 

6. 10 M Ammonium acetate. 

7. Absolute ethanol. 

8. 70% Ethanol. 

2.2. Restriction Enzyme Digestion 

1. Taql(10U/\lL). 

2. S.vpI(10U/uL). 

3. Other restriction enzymes. 

2.3. Circularization and Cleavage or Denaturation 

1. 5X ligation buffer (supplied with T4 DNA ligase). 

2. T4 DNA ligase (IU/ixL). 

3. T4 RNA ligase (4 units/uL). 

4. 15 uM Hexaminecobalt chloride. 

5. Phenol: CHCl 3 :isoamyl alcohol (25:24:1). 

6. 3 M Sodium acetate, pH 7.0. 

7. Absolute ethanol. 

8. 70% Ethanol. 

2.4. Inverse PCR 

1. 10X PCR buffer: 100 mM Tris-HCl, pH 8.3, 500 mM KC1, 15 mM MgCl 2 , 0.01%(w/v) 
gelatin. 

2. 15mMMgCl 2 . 

3. Deoxyoligonucleotides were synthesized on an Applied Biosystems (Foster City, CA) 
392B DNA synthesizer and purified by OPC column from the same company. 

4. Taq DNA polymerase. 

3. Methods 

3.1. Genomic DNA Isolation 

1. For culture cells, trypsinize, wash twice with PBS and resuspend in cold PBS. Use approx 
1 mL per 2 x 10 7 cells. For tissues, freeze quickly in liquid nitrogen and mince into small 
pieces with scalpel and place in approx 5 volumes of PBS. Work quickly to prevent deg- 
radation by nucleases. 

2. Add 1 mL of proteinase K (10 mg/mL) per 10 mL PBS. 

3. Immediately add equal volume of Premix I. Mix gently by inversion. 

4. Incubate at 50 C C overnight. 

5. Extract with equal volume of phenol/chloroform/isoamyl alcohol 25:24:1. 

6. Transfer aqueous phase to a new tube. Re-extract the organic phase with an equal volume 
of N25QTKJE,. Pool the aqueous phases. 

7. Ethanol precipitate with one quarter volume 10 M ammonium acetate and 2 volumes 
ethanol. The DNA should precipitate immediately. Spool the DNA out with a Pasteur 
pipet, or spin for 2 min. 

8. Wash DNA pellet with 70% ethanol, then dry briefly (do not overdry or DNA will be 
difficult to dissolve) and resuspend in 25 (xL distilled water. 



IPCR: Genomic DNA Cloning 305 

3.2. Restriction Enzyme Digestion 

1 . Add components to the genomic DNA on ice in the following order: 
1 OX reaction buffer 12.5 (xL 

Nuclear-free water 95 (xL 

Restriction enzymes 5 [xL 

2. Incubation at 37 C C for 2 h. 

3. Stop the reaction with 12.5 uL of 0.2 M EDTA and 200 u.L sterile H 2 0. 

4. Concentrate and purify the sample with GeneClean. Resuspend the DNA in 100-200 (xL 
of sterile H,0. 

5. Estimate the DNA concentration by comparing the ethidium bromide fluorescent inten- 
sity of the sample with that of a series of DNA standards on a sheet of plastic wrap. Dot 
1-5 (xL of sample onto plastic wrap on a UV transilluminator. Also dot with 5 |xL of 
DNA standards. Add an equal volume of TE buffer containing 2 [xg/mL of ethidium 
bromide, mix by repipetting up and down. Use proper UV shielding for exposed skin 
and eyes. 

3.3. Circularization and Cleavage (see Notes 1-4) 

1. Set up the circularization reaction mix containing the following components: 100 |xL 
(100 ng DNA, see Note 1) of the purified sample, 25 [xL of 5X ligation buffer, and 6 (xL 
of T4 DNA ligase. Finally, add 2 fxL of T4 RNA ligase or 15 uL of 15 (xM hexaminecobalt 
chloride (see Note 5). 

2. Incubate at 18°C for 16 h. 

3. Boil the ligated circular DNA for 2-3 min in distilled water or digest with an appropriate 
restriction enzyme to reopen circularized DNA (see Notes 2-4). 

4. Purify the DNA sample with GeneClean as described in step 6 in Subheading 3.2. or 
extract with water-saturated phenol/CHC13 and then precipitate with ethanol. 

3.4. Inverse PCR 

1. Add 1/10 of the purified cDNA to 100 [xL of amplification mix: 
1 OX PCR buffer 10 uL 

15mMMgCl 2 10 uL 

dNTP mix (2 . 5 mM of each) 1 uL 

5'-primer (10 pmole/(xL) 10 (xL 

3'-primer (10 pmole/fxL) 10 u.L 

cDNA 10 uL 

Nuclease-free H 2 39.5 (xL 

Taq DNA polymerase 2.5 (lyiL) 0.5 [xL 

2. Cap and vortex the tubes to mix. Spin briefly in a microfuge. Cover each reaction with a 
few drops of light mineral oil to prevent evaporation. 

3. Put a drop of mineral oil into each well of the thermal cycler block that will hold a tube. 
Load the reaction tubes. 

4. Amplify by PCR using the following cycle profile: 
25 cycles 94°C, 1 min (denaturation) 

65°C, 2 min (annealing) 
72°C, 4 min (elongation) 



306 Jong et al. 

4. Notes 

1. The principle of IPCR is simple and the protocol is straightforward. The trickiest part is 
to obtain the targeted DN A after ligation, because of the high randomness of the ligation 
reaction. For maximum efficiency of intramolecular ligation, a low DNA concentration 
should be used in the ligation mix. High density of DNA may enhance the level of hetero- 
geneous ligation, which creates nonspecific amplification. 

2. Cleavage or denaturation of circularized double-stranded DNA is important because cir- 
cular double-stranded DNA has a tendency to form supercoil which is a poor template for 
PCR (17). Circularized double-stranded DNA is only good for amplification of a short 
DNA fragment. 

3. The following three ways can be considered to introduce nicks in circularized DNA. 
Boiling is a simple and common way. Because of the unusual secondary structure of 
some circular double-stranded DNA, sometimes this method is not sufficient in nicking 
and denaturing circular double-stranded DNA. A second method is selected restriction 
enzyme digestion. The ideal restriction site is located within the known sequence 
region of DNA. In most cases, however, it is difficult to make the right choice of a 
restriction enzyme because the restriction pattern in the unidentified region of DNA is 
unknown. If an appropriate enzyme is not available, EDTA-oligonucleotide-directed 
specific cleavage may be tried (18). Oligonucleotide linked to EDTA-Fe at T can bind 
specifically to double-stranded DNA by triple-helix formation and produce double- 
stranded cleavage at the binding site. 

4. Alkaline denaturation has been successfully used to prepare plasmid DNA templates for 
PCR and DNA sequencing (19). This method should be feasible in denaturing circular- 
ized double-stranded cDNA. 

5. Inclusion of T4 RNA ligase or hexaminecobalt chloride can enhance the efficiency of 
blunt-end ligation of double-strand DNA catalyzed by T4 DNA ligase (20). 

References 

1. Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse 
polymerase chain reaction. Genetics 120, 621-625. 

2. Triglia, T., Peterson, M. G, and Kemp, D. J. (1988) A procedure for in vitro amplification of 
DNA segments that lie outside the boundaries of known sequences. Nucl. Acids Res. 16, 8186. 

3. Huang, S.-H., Hu, Y. Y., Wu, C.-H., and Holcenberg, J. (1990) A simple method for direct 
cloning cDNA sequence that flanks a region of known sequence from total RNA by 
applying the inverse polymerase chain reaction. Nucl. Acids Res. 18, 1922. 

4. Li, Z. H., Liu, D. P., and Liang, C. C. (1999) Modified inverse PCR method for cloning the 
flanking sequences from human cell pools. Biotechniques 27, 660-662. 

5. Triglia, T. (2000) Inverse PCR (IPCR) for obtaining promoter sequence. Meth. Mol. Biol. 
130, 79-83. 

6. Yoshitomo-Nakagawa, K., Muramatsu, M., and Sugano, S. (1997) Cloning of the pro- 
moter regions of mouse TGF-beta receptor genes by inverse PCR with highly overlapped 
primers. DNA Res. 4, 73-75. 

7. Pham, H. S., Kiuchi, A., and Tabuchi, K. (1999) Methods for rapid cloning and detection 
for sequencing of cloned inverse PCR-generated DNA fragments adjacent to known 
sequences in bacterial chromosomes. Microbiol. Immunol. 43, 829-836. 

8. Martin, V. J. and Mohn, W. W. (1999) An alternative inverse PCR (IPCR) method to amplify 
DNA sequences flanking Tn5 transposon insertions. J. Microbiol. Meth. 35, 163-166. 



IPCR: Genomic DNA Cloning 307 

9. Gama, L. and Breitwieser, G. E. (1999) Generation of epitope-tagged proteins by inverse 
PCR mutagenesis. Biotechniques 26, 814-816. 

10. Xu, Y. and Gong, Z. (1999) Adaptation of inverse PCR to generate an internal deletion. 
Biotechniques 26, 639-641. 

11. Pues, H., Holz, B., and Weinhold, E. (1997) Construction of a deletion library using a 
mixture of 5'-truncated primers for inverse PCR (IPCR). Nucl. Acids Res. 25, 1303-1304. 

12. Eisinger, D. P. and Trumpower, B. L. (1997) Long-inverse PCR to generate regional pep- 
tide libraries by codon mutagenesis. Biotechniques 22, 250-254. 

13. Li, Z. X., Yoshimura, S., Kobayashi, T., and Akane, A. (1998) Allele-specific, inverse- 
PCR amplification for genotyping MN blood group. Biotechniques 25, 358-360, 362. 

14. Raponi, M., Dawes, I. W., and Arndt, G. M. (2000) Characterization of flanking sequences 
using long inverse PCR. Biotechniques 28, 838-844. 

15. Pang, K. M. and Knecht, D. A. (1997) Partial inverse PCR: a technique for cloning flank- 
ing sequences. Biotechniques 22, 1046-1048. 

16. Wang, P., Ka-Wai, H. E., Chiu, J., and Lo, S.J. (2001) Analysis of integrated hepatitis B 
virus DNA and flanking cellular sequence by inverse polymerase chain reaction. ./. Virol. 
Meth. 92, 83-90. 

17. Moon, I. S. and Krause, M. O. (1991) Common RNA polymerase I, II, and III upstream 
elements in mouse 7SK gene locus revealed by the inverse polymerase chain reaction. 
DNA Cell Biol. 10, 23-32. 

18. Strobel, A. S and Dervan, P. B. (1990) Site-specific cleavage of a yeast chromosome by 
oligonucleotide-directed triplex formation. Science 249, 73-75. 

19. Cusi, M. G., Cioe, L., and Rovera, G. (1992) PCR amplification of GC-rich templates 
containing palindromic sequences using initial alkali denaturation. Biotechniques 12, 
502-504. 

20. Sugino, A., Goodman, H. M., Heynecker, H. L., Shine, J., Boyer, H. W., and Cozzarelli, 
N. R. (1977) Interaction of bacteriophage T4 RNA and DNA ligase in joining of duplex 
DNA at base-paired ends. J. Biol. Chem. 252, 3987. 



32 

Gene Cloning and Expression Profiling 
by Rapid Amplification of Gene Inserts 
with Universal Vector Primers 

Sheng-He Huang, Hua-Yang Wu, and Ambrose Y. Jong 
1. Introduction 

Isolation of a full-length gene and analysis of expression profiling are fundamental 
and challenging in the current molecular biology. A great deal of effort is needed to 
detect unknown gene sequences by screening cDNA or genomic libraries by nucleic 
acid or protein probes. As the complete genome sequences of many organisms have 
been reported, this has raised the most challenging issue that how global gene expres- 
sion patterns can be detected. Recently, various PCR methods have been developed 
for cloning of unknown genes and analysis of expression profiling (1-10). Several 
PCR-based strategies such as iAFLP (introduced amplified fragment length polymor- 
phism) and TOGA (total gene-expression analysis) are currently available for global 
gene-expression analysis (8-10). An outline of gene cloning and expression profiling 
by anchored-PCR with vector primers is illustrated in Fig. IB. In this chapter, we focus 
on how to use a gene library and anchored-PCR for cloning unknown gene sequences 
(see Fig. 1A). Friedmann et al. (1) first used PCR to screen Xgtll library with two 
gene-specific primers. This protocol can be effectively used to isolate a particular 
DNA fragment between two specific primers or to generate nucleic acid probe from 
cDNA libraries. The unknown sequences flanking the fragment between the two spe- 
cific primers can not be amplified by this method. 

Anchored-PCR or single-specific-primer PCR (2,3) and inverse PCR (4) have been 
adapted to cloning of full length cDNAs with the knowledge of a small stretch of 
sequence within the gene. Both methods start from mRNA and are good for cDNA 
cloning when a cDNA library is not available. Cloning of full-length cDNA is usually 
far more difficult than any other recombinant DNA work because the multiple sequen- 
tial enzymatic reactions often result in low yield and truncated clones (11). Shyamala 
and Ames (3) extended the use of anchored PCR to amplify unknown DNA sequences 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

309 



310 



Huang, Wu, and Jong 




(RAGE) A 



^r linearize \ 

I* 3'-VP * 



B (TOGA) 



T3 Promoter 



PCR1 




1 



transcribe 



a i cr 



ssDNA wm 

PCR2 

dsDNA ^K 



I 



PCR3 



\ / 



— ► 

5P, 


X cDNA synthesis 


5P 2 N, 


a™ cDNA 

1 PCRl 3P 


5P 2 N M 


■ !/4cDNA 

1 * _ 

y PCR2 3PF 


^wil 


-™ 1/256 F-cDNA 



Fig. 1 . The scheme for Gene Cloning and Expression Profiling by amplification of gene 
inserts from a library. (A) Gene cloning by rapid amplification of gene ends (RAGE). If a 
plasmid library is used, a restriction enzyme rarely within gene inserts such as NotI may be 
selected to linearize the recombinant DNA. A phage library can be directly used for PCR. Here 
is an example for cloning cDNA ends from a phage library (Xgtl 1). 5'-VP is 'kglll forward 
primer (5'-GACTCCTGGAGCCCG-3'). 3'-VP: Xgtll reverse primer (5'-GGTAGCGACC- 
GGCGC-3'). 5'-GSP (gene specific primer): 5ASPR with £coRl restriction site (5'-AGA- 
CTGAATTCGGTACCGGCGGTACTATCGCTTCC-3'). 3'-GSP: 3ASPB containing BamWX 
site (5'-CTGATGGATCCTGGCAGTGGCTGGACGC-3'). (B) Expression profiling by TOGA. 
Poly(A) RNA isolation, cDNA synthesis and library construction were carried out as described 
previously (10). The 3' Mspl-Notl fragments were directionally cloned into Clal-Notl cleaved 
plasmid pBC SK+ (Stratagene) in an orientation antisense to its T3 promoter. After cleavage 
with Mspl to linearize insert-containing plasmids, antisense transcripts were produced with T3 
RNA polymerase. The cRNAs were reverse transcribed into cDNA by using 5P! [a 5' vector 
primer including a 4-nt restriction endonuclease cleavage site (4-nt REC) derived from the 
ligation of the vector with the 5' insert]. PCRl was performed with primers SPiNj [a 5' vector 
primer plus the 4-nt REC and the first adjacent nucleotide (N1=A,G,T,C) of the insert at the 
3' end ] and 3P (a 3' vector primer). This step subdivides the cDNA species into four pools. 
PCR2 was carried out with 3PF (a fluorescent 3' vector primer) and 5P 2 Ni_ 4 [(a 5' vector primer 
containing the 4-nt REC and four 3' degenerate nucleotides (N^, N=A,C,G,T), which are the 
adjacent nucleotides of the insert]. This amplification subdivides the input species into 256 
subpools for electrophoretic resolution. 



RA GE with Universal Primers 3 1 1 

from genome on the basis of using a short stretch of known sequence for designing a 
gene-specific primer. We developed a much simpler method to isolate full-length 
cDNA and flanking genomic DNA from gene libraries by anchored-PCR. The prin- 
ciple of this technique is schematically depicted in Fig. 1A. Briefly, two ends of a 
gene were amplified by two vector primers (VP) complementary to the vector 
sequences flanking the polylinker region and two gene-specific primers (GSP) comple- 
mentary to 5' and 3' parts of the known gene sequence. 

We used the yeast gene coding for asparaginase II (12) as a model to verify this 
method. Two ends of the yeast asparaginase II gene (12) were amplified from Kgtl 1 
yeast genomic library by this method. The size of the full-length gene is 1 .7 kb and 
there is 353 bp overlapping sequence between the two GSPs (5ASPR and 3ASPB). 
The 3' gene end (from 5ASPR to the extreme 3' end of the gene) and the 5' gene end 
(from 3ASPB to the very beginning of 5' part of the gene) are 1.15 and 0.9 kbs, respec- 
tively. Because there may be two different orientations for each insert in the gene 
library, four reactions are performed for the amplication of two gene ends in the first 
round of PCR. Fig. 2 shows that there was only one orientation for the asparagi- 
nase II gene clone and the expected sizes of the two amplified gene ends were obtained. 
The same size fragment was amplified from the gene library and the two purified gene 
ends by using the two GSPs as primers. The gel-purified PCR fragments were 
sequenced and the sequence data were consistent with the literature (12). 

We amplified and isolated 5'-cDNA fragment of 5 -hydroxy tryptamine 2 (5-HT2) 
receptor from lambda SWAJ-2 mouse brain cDNA library by this technique. 5'-cDNA 
region of 5-HT2 receptor was successfully amplified from XSWAJ-2 mouse brain 
cDNA library with a GSP (5'-TTCTGCCTGAGACTAAAAAGGGTTAAGCCCTTA- 
TGATGGCA-3') and a VP (Xba 1-T15 adaptor primer: 5'-GTCGACTCTTAGAT-3') (5). 

2. Materials 

1. Taq DNA polymerase (Perkin/Elmer, Foster City, CA). 

2. dNTP: 10 mM of each (Perkin/Elmer). 

3. Yeast genomic library (ClonTech, Palo Alto, CA). See Note 1. 

4. >^SWAJ-2 mouse brain cDNA library (ClonTech). 

5. 5' and 3' Xgtl 1 primers (ClonTech). 

6. Geneclean™ kits (BiolOl, La Jolla, CA). 

7. Sequenase kits (United States Biochemical Corporation, Cleveland, OH). 

8. 10X Taq DNA polymerase buffer: 67 mM Tris-HCl, pH 8.8, 6.7 mM MgCl 2 , 170 jig/mL 
bovine serum albumin (BSA), 16.6 mM ammonium sulfate (New England Biolab, 
Beverly, MA) was prepared as described (2; see Note 4). 

9. Oligodeoxyribonucleotides were synthesized on an Applied Biosystems 380B DNA 
synthesizer and purified by OPEC column from the same company. 

10. Programmable Thermal Controller. 

3. Methods 

The following conditions were used to amplify the ends of the yeast asparaginase II 
gene from a Xgtl 1 yeast genomic library. 



312 



Huang, Wu, and Jong 



r\o i 


m 


4.0- 
3,0- 




2P- 
1.6- 


~~ i 


1$M 


i - | 


05- 


mm* 



1 



3 4 5 6 7 8 



Fig. 2. Gel pattern of PCR products. Fragments amplified from the purified 5' (lane 6) and 3' 
(lane 7) gene ends and A.gtl 1 yeast genomic library (lane 2, 3, 4, 5, and 8) were resolved in 
1.5% agarose gel and stained with ethidium bromide. Lane 1 was 1 Kb ladder DNA marker 
(BRL). There were five pairs of primers used in PCR: 1) 5'-VP and 3'-GSP (lane 2); 2) 3'-VP 
and 3'-GSP (lane 3); 3) 5'-VP and 5'-GSP (lane 4); 4) 3'-VP and 5'-GSP (lane 5), and 5) 5'-GSP 
and 3'-GSP (lane 6,7 and 8). 



3. 1. PCR#1 (see Notes 2 and 3) 

1 . In order to enhance the specificity of amplification, asymmetric PCR was carried out in 
50 (xL of reaction mixture containing 1 (xL aliquot of Xgtl 1 yeast genomic library (about 
1 x 10 7 PFU), 2.5 pmol vector primer (VP), 50 pmol GSP, 5 u.L of dimethylsulfoxide, 
5 [xL of 10X Taq DNA polymerase buffer (2), 1.5 mM of each dNTP, and 2.5 U of Taq 
DNA polymerase. Before adding the enzyme, the PCR cocktail was heated at 94°C for 
3 min to disrupt the phage particles. 

2. PCR parameters were: 35 cycles at 94°C for 1 min, 48°C for 30 s, and 72°C for 8 min. 
The major product, single-strand DNA, can be used for sequencing and Southern blotting 
analyses. 

3.2. PCR#2 

1 . Dilute the first PCR product at 1 : 10 in H 2 0. 

2. Amplify 1-jxL aliquots as described in PCR#1, with the exception of using equal amounts 
(50 pmol) of the VP and GSP. The selected dsDNA fragments are available for cloning, 
which can be facilitated by incorporating restriction sites in the primers. 



RAGE with Universal Primers 313 

3.3. PCR#3 

PCR 3 is used to test the products of PCR 2 because amplification of the two GSPs 
should produce the same fragment from a gene library and the two purified gene ends. 

1. Assemble a PCR with 1 (xL of 1:10 diluted PCR2 product or l-(xL aliquot of gene library 
were used in the third round of PCR using two GSPs (50 pmol each). 

2. Amplify for 35 cycles at 94°C for 30 s, 48°C for 30 s and 72°C for 4 min. 

3. Purify DNA fragments from PCR#1, 2, and 3 with GeneClean™ (BIO 101 Inc.) from 
agarose gel and then sequence by the dideoxy chain termination method (13) with 
Sequenase (USB) with the aid of NP40 (14) (see Notes 4 and 5). 

4. Notes 

1. The successful isolation and detection of gene inserts from cDNA or genomic libraries is 
dependent on the quality of the library. There is a big difference between the primary and 
the amplified libraries because different recombinant clones may grow at very different 
rates, resulting in unequal distribution of the recombinants in the amplified libraries (19). It 
may be better to use the primary library for PCR amplification. If a short stretch of known 
DNA sequence (more than 100 bp) is available, it is easy to test the quality of the library by 
PCR with two GSPs. In case the library is not good, an alternative strategy is to use inverse 
or anchored PCR to isolate or detect gene inserts from genomic DNA or self-made cDNA. 

2. The most obvious and common problem for PCR with single-sided specificity is nonspe- 
cific amplification of DNA fragments without significant homology with the gene of 
interest (15,17). In this chapter we found two modifications that improve specific ampli- 
fication. At first, we used PCR buffer from Perkin Elmer Cetus and failed to amplify 
specific products for yeast asparaginase II gene. It appears that the Taq DNA polymerase 
buffer from New England Biolab is much better for PCR with single-specific primer. 
Second, asymmetric PCR with a relatively large amount of GSP was performed in the 
first round of PCR in order to enhance the specificity of amplification. 

3. There is a limitation for DNA amplification with degenerate primers based on the highly 
conserved regions of a protein from other species or limited amino acid sequence data 
because degeneracy of primers can create more problems with nonspecific amplification. 
This limit may be reduced by incorporation of deoxyinosine into wobble positions of 
degenerate oligonucleotides (IS). 

4. This method can be used for directional genome walking from known into unknown flank- 
ing regions of the chromosomal DNA with genomic libraries. PCR amplification of large 
DNA fragments (up to 35 kb) has been achieved by the combination of a high level of an 
exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaql, with a 
very low concentration of a thermostable DNA polymerase exhibiting a 3'-exonuclease 
activity (Pfu, Vent, or Deep Vent) (18). 

5. The two gene ends with overlapping sequence can be simply linked by two ways. First, 
ssDNAs of the two gene ends from PCR1 can be annealed and end filled by Klenow in 
the presence of random primers. Second, when the sequence information is obtained 
from the two gene ends, a full length cDNA or gene can be amplified by two specific 
primers that represent the sequences at the extreme 3' and 5'-ends of the gene or cDNA. 

Acknowledgments 

The authors would like to acknowledge Dr. J. Holcenberg for his generous support. 
We especially thank C.-H. Wu and B. Cai for their technical assistance. This work was 
supported by the T.J. Martell Foundation. 



314 Huang, Wu, and Jong 

References 

1. Friedmann, K. D., Rosen, N. L., Newman, P. J., and Montgomery, R. R. (1988) Enzymatic 
amplification of specific cDNA inserts from >^gtl 1 libraries. Nucl. Acids Res. 16, 8718. 

2. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full-length 
cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide 
primer. Proc. Natl. Acad. Sci. USA 85, 998-9002. 

3. Shyamala, V. and Ames, G. F.-L. (1989) Genome walking by single-specific -primer poly- 
merase chain reaction: SSP-PCR. Gene 84, 1-8. 

4. Huang, S. H., Hu, Y. Y., Wu, C. H., and Holcenberg, J. (1990) A simple method for direct 
cloning cDNA sequence that flanks a region of known sequence from total RN A by apply- 
ing the inverse polymerase chain reaction. Nucl. Acids Res. 18, 922. 

5. Huang, S.-H., Jong, A. Y., Yang, W., and Holcenberg, J. (1993) Amplification of gene 
ends from gene libraries by polymerase chain reaction with single-sided specificity. Meth. 
Mol. Biol. 15, 357-363. 

6. Tsurui, H., Hara, E., Oda, K., Suyama, A., Nakada, S., and Wada, A. (1990) A rapid and 
efficient cloning method with a solid-phase DNA probe: application for cloning the 
5'-flanking region of the gene encoding human fibronectin. Gene 88, 233-239. 

7. Cormack, R. S. and Somssich, I. E. (1997) Rapid amplification genomic ends (RAGE) as 
a simple method to clone flanking genomic DNA. Gene 194, 273-276. 

8. Kawamoto, S., Ohnishi, T., Kita, H., Chisaka, O., and Okubo, K. (1999) Expression pro- 
filing by iAFLP: A PCR-based method for genome-wide gene expression profiling. 
Genome Res. 9, 1305-1312. 

9. Wang, A., Pierce, A., Judson-Kremer, K., Gaddis, S., Aldaz, C. M., Johnson, D. G., and 
MacLeod, M. C. (1999) Rapid analysis of gene expression (RAGE) facilitates universal 
expression profiling. Nucl. Acids Res. 27, 4609-4618. 

10. Sutcliffe, J. G., Foye, P. E., Erlander, M. G., Hilbush, B. S., Bodzin, L. J., Durham, J. T., 
and Hasel, K. W. (2000) TOGA: an automated parsing technology for analyzing expres- 
sion of nearly all genes. Proc Natl Acad Sci USA 97, 1976-1981. 

11. Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T., and Arai, K. (1987) 
High-efficiency cloning of full-length cDNA: construction and screening of cDNA 
expression libraries for mammalian cells. Meth. Enzymol. 154, 3-28. 

12. Kim, K. W., Kamerud, J. Q., Livingston, D. M., and Roon, R. J. (1988) Asparaginase II 
of Saccharomyces cerevisiae: characterization of the ASP3 gene. ./. Biol. Chem. 263, 
11,948-11,953. 

13. Sanger, F., Nickler, S., and Coulson, A. R. (1977) DNA sequencing with chain-terminat- 
ing inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 

14. Bachmann, B., Lucke, W., and Hunsmann, G. (1990) Improvement of PCR amplified DNA 
sequencing with the aid of detergents. Nucl. Acids Res. 18, 1309. 

15. Patil, R. V. and Dekker, E. E. (1990) PCR amplification of an Escherichia coli gene using 
mixed primers containing deoxyinosine at ambiguous positions in degenerate amino acid 
codons. Nucl. Acids Res. 18, 3080. 

16. Frohman, M. A. (1990) RACE: rapid amplification of cDNA Ends, in PCR Protocols: A 
Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, 
T. J., eds.), Academic, San Diego, CA, pp. 28-38 

17. Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., and Davis, M. M. (1989) Polymerase 
chain reaction with single-sided specificity: analysis of T cell receptor 6 chain. Science 
243,217-220. 

18. Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high 
yield from ^bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216-2220. 

19. Frischauf, A.-M. (1987) Construction and characterization of a genomic library in X. Meth. 
Enzymol. 152, 190-199. 



33 



The Isolation of DNA Sequences Flanking Tn5 
Transposon Insertions by Inverse PCR 

Vincent J. J. Martin and William W. Mohn 
1. Introduction 

Because of its versatility, the Tn5 transposon has become a powerful tool in the 
classical genetic studies of Gram-negative bacteria. The Tn5 transposon is functional 
in a broad range of Gram-negative bacteria and transposes at a high frequency with 
low specificity of insertion (7 ,2), allowing it to insert in a large number of locations in 
bacterial genomes. The initial use of the Tn5 transposon was in the identification of 
disrupted genes by insertion muatgenesis and phenotypic screening, however, deriva- 
tives of this mobile element were later developed for use in reporter gene fusion (3) 
and promoter probing experiments (4). 

The characterization of the region of Tn5 insertion in a genome requires the isola- 
tion of DNA flanking the insertion. This was conventionally accomplished by cloning 
the mutagenized DNA fragment containing both upstream and downstream regions 
relative to the insertion, along with the transposon (5). To isolate the flanking DNA, 
clone libraries were constructed from the Tn5 mutated DNA and screened on selective 
medium containing kanamycin. This approach for isolating the DNA of interest is 
time-consuming and labor-intensive because it requires the production and screening 
of genomic clone libraries and subcloning of the positive clones prior to sequencing. 

Several methods were developed to reduce the time required to isolate and sequence 
DNA regions flanking transposon insertions. These methods include vector-mediated 
rescue of mutations (6,7) and polymerase chain reaction (PCR)-based methods such as 
linker-mediated, single-primer, anchored, repetitive extragenic palindrome (REP) and 
inverse PCR (8-14). Because of their ease, these methods also allow for the simulta- 
neous characterization and isolation of large numbers of clones from a mutation library. 

In this chapter, we describe a simple and reliable experimental procedure to clone 
DNA flanking a Tn5 transposon by inverse PCR (IPCR). The IPCR method is based 
on the initial report from Rich and Willis (12) and incorporates the improvements 
from Martin and Mohn (13) and Huang et al. (14). The basic strategy for the Tn5 IPCR 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

315 



3 1 6 Martin and Mohn 

is shown schematically in Fig. 1. The technique requires three basic steps: 1) the 
restriction enzyme digestion of Tn5-tagged genomic DNA, 2) circularization of the 
fragments by intra-molecular ligation, and 3) amplification with PCR primers designed 
to anneal to the Tn5 transposon. In a first approach, the choice of restriction endonu- 
clease to use in IPCR template preparation is established by Southern blot analysis 
using an asymmetric probe, which hybridizes to a small region of both the left and 
right side IS50 regions as well as the genes encoding antibiotic resistance (see Fig. 2). 
For enzymes that do not cut the transposon, a single band will appear in the Southern 
blot corresponding to 5.8 kb + the size of flanking DNA from both ends. In using 
enzymes that cut once within the transposon, two bands will appear from the Southern 
blot. The thick band corresponds to the left side of the Tn5, which is complementary to 
the entire probe and the light band to the right side of the transposon, which is comple- 
mentary to only one end of the probe (see Fig. 2). The size of the IPCR products from 
templates prepared from each of the restriction endonucleases tested can be inferred 
from the size of the bands in the Southern blot analysis. Although knowing the 
expected size of the IPCR product allows a certain level of confidence in the results, it 
is not always necessary. Huang et al. (14) reported on a method using a nested PCR 
primer (IR2, Table 1), which allowed discrimination of target from nonspecific 
products without resorting to Southern blot analysis. 

2. Materials 

2. 1. Southern Blot Analysis 

A list of general materials and methods used for genomic DNA preparations, South- 
ern blot analysis of genomic DNA and radioisotope labeling of DNA probes is found 
in Sambrook and Russell (15). Additional requirements follow. 

1. Genomic DNA (2.5-5 ug per restriction endonuclease tested) prepared from Tn5 
transposon mutant strain(s) of interest. 

2. A selection of restriction endonucleases that do not cut the Tn5 transposon or that cut the 
transposon only once (see Note 1). Restriction endonucleases are normally supplied with 
their appropriate buffers. 

Examples of enzymes that do not cut the Tn5 transposon are: Aatll, Aflll, Apal*, 
ApalA, Asel, AvrIL, BsrGl, CM*, Dial**, EcoRl, EcoRV**, Kpnl, MM, Ndel, Nsil, 
Pmll**, Pvul, Sad*, Seal**, Spel, Stul* ** and Xbal*(for * and **, see Note 1). 
Enzymes that cut the Tn5 transposon only once are: BamHl, Sail, Smal**/Xmal, 
and Sphl. 

3. Hindlll-BamHl 1861-bp probe from the Tn5 transposon (see Fig. 2) labeled with [a 32 P]- 
dCTP by nick translation (labeling kit sold by Life Technologies) (see Notes 2 and 3). 

2.2. Preparation of Inverse PCR Template 

1. Sterile, redistilled water. 

2. Genomic DNA (1 u,g) isolated from Tn5 mutant strain(s) of interest. 

3. Restriction endonuclease(s) selected from the results of the Southern blot. 

4. TE saturated phenol:chloroform:isoamyl alcohol (25:24:1). 

5. Absolute ethanol. 

6. 70 % ethanol. 



CO 

■—4. 

N 



J— k 



'"I g 



2000 a. 



Tn5(5818bp) 

S 

£ ^ 3000 a 

X « IB) 

-n — 



UTn5 IR1 IR2 



BamVtl digestion 
and ligation 




XmalJn5L 

-► 
SphlTnSL SallTnSL ' 

I nptll fesfe 



T 



BL 



SphlTnBU IR1 UTnS 



EcoRl Sphl 

■UL 



^^ XmalTn5 



SphlTnSR 



SallTnSR BR 



Ecoltl digestion 
and ligation 



i 



EcoRl 





SphlTnS 



SphlTnSR 

Sphl Sphl 



|R2 SphlTn5R 

Fig. 1 . Schematic diagram showing the strategy to amplify DNA sequences flanking the Tn5 transposon 
by IPCR. Black bars represent unknown DNA sequences flanking a Tn5 insertion; gray bars represent the 
left (IS50 L ) and right (IS50 R ) insertion sequences. Small arrows symbolize the location of the PCR primers 
described in Table 1. Antibiotic resistance genes are nptll, neomycin/kanamycin; ble, bleomycin ; str, 
streptomycin. 



318 



Martin and Mohn 



JScoRI BamHl 

I I 



Tn5 insertion in chromosome 



Hindlll BamHl Hindlll 

I 



Probe 

Southern blot analysis 
of mutated DNA 




£coRI BamHl 



Both sides of the 
Tn5 transposon 



EcoRl BamHl 

I I 



Right side of the 
Tn5 transposon 

Left side of the 
Tn5 transposon 



Fig. 2. Schematic diagram of the Southern blot analysis strategy used to predict the size of the 
IPCR products [based on Mitchell and Smit, 1990 (17)]. The gray bars represent the left and right 
insertion sequences IS50, which are inverted terminal repeats in the Tn5 transposon. The light 
band in the blot represents hybridization of the Hindlll-BamHl DNA probe to the right side of the 
transposon and the thick band represents hybridization to the left side of the transposon. 



7. 3 M cold sodium acetate pH 7.0. 

8. T4 DNA ligase (400 U/uL) (New England Biolabs). 

10. 10X ligation buffer containing 10 mM ATP (supplied with T4 DNA ligase). 

2.3. Inverse PCR 

1. Taq and Pwo DNA polymerase enzyme mix (Expand High Fidelity PCR System, Roche 
Molecular Biochemical) (see Note 4). 

10X PCR buffer (Expand HF buffer supplied with the PCR system). 
Sterile 25 mM MgCl 2 stock solution (supplied with the PCR system). 
PCR grade nucleotide mix containing 10 mM of each dNTP. 
Deoxyoligonucleotide primers (Table 1). 

Light mineral oil (may not be required if thermocycler uses a heated lid). 
pRZ705, pSUP2021, or any other plasmid containing a Tn5 insertion to be used as posi- 
tive control template for the IPCR reaction (optional). 

3. Methods 

3.1. Preparation of Inverse PCR Template 

Based on the results of the Southern blot analysis, select an endonuclease enzyme(s) to 
use for preparing the IPCR template(s). The enzymes should produce an IPCR template 
that will yield IPCR products of < 5 kb (see Note 4). The predicted size of the IPCR 



1. 



Table 1 

List of Primers for Tn5 IPCR 



Name 



DNA Sequence 



Length 
(bp) 



Restriction 
enzyme 



Region of 
Tn5 transposon" 



Reference 



Co 

— I 



UTn5 

IR1 

XmaITn5R 

XmaITn5L 

XmaITn5U ft 

SalITn5R 

SalITn5L 

SallTnSU 6 

SphITn5R 

SphITn5L 

SphITn5U b 

BR 

BL 

IR2 



5'-GGTTCCGTTCAGGACGCTAC-3' 

5'-GAGCAGAAGTTATCATGAACG-3' 

5'-AGGCAGCAGCTGAACCAA-3' 

5-GCCGGCTGGATGATCCTC-3' 

5'-CGCCCGGGTCACATGGAAGTCAGATCCTG-3' 

5'-ATGCCTGCAAGCAATTGC-3' 

5'-AACCAGCAGCGGCTATCC-3' 

5'-TGGGTCGACTCACATGGAAGTCAGATCCTG-3' 

5'-ATTCGGCAAGCAGGCATC-3' 

5'- TGGACGAAGAGCATCAGG-3' 

5'-CCGCATGCTCACATGGAAGTCAGATCCTG-3' 

5'- CATTCCTGTAGCGGATGGAGATC -3' 

5 - GGGGACCTTGCACAGATAGC -3' 

5'-CGGGATCCTCACATGGAAGTCAGATCCTG-3' 



20 


All enzymes 


37- 


-18 and 5782-5801 


(12) 


21 


All enzymes 


107- 


-87 and 5712-5732 


(14) 


18 


Xmal 




2556-2539 


(13) 


18 


Xmal 




2462-2479 


This study 


29 


Xmal 


70- 


-50 and 5749-5769 


This study 


18 


Sail 




2739-2722 


(13) 


18 


Sail 




2614-2031 


This study 


30 


Sail 


70- 


-50 and 5749-5769 


This study 


18 


Sphl 




2135-2118 


This study 


18 


Sphl 




2023-2040 


This study 


29 


Sphl 


70- 


-50 and 5749-5769 


This study 


23 


BamHl 




3134-3112 


(14) 


20 


BamHl 




3007-3026 


(14) 


29 


BamHl 


70- 


-50 and 5749-5769 


(14) 



"Based on the complete sequence of Escherichia coli transposon Tn5 from GenBank accession* U00004 
'The annealing regions of these primers are based on the IR2 primer of Huang et al. (14). 



320 Martin and Mohn 

product is calculated by subtracting the size of the Tn5 DNA fragment flanking the target 
sequence from the size of the band that hybridized to the probe (see Fig. 2). 

2. Digest 1 u.g of the genomic DNA. 

3. Heat inactivate the endonuclease(s) at 65°C for 20 min and centrifuge briefly to collect 
condensate. 

4. Extract the digested DNA with an equal volume of TE saturated phenol:chloroform:iso- 
amyl alcohol (25:24:1). 

5. Precipitate the DNA with 1/10 vol cold sodium acetate and 2 vol of absolute ethanol. 
Centrifuge and wash the DNA pellet with 70% ethanol (15). Suspend the DNA in 180 uE 
of sterile H 2 0. 

6. Set up the template circularization reaction with the 180 uE of cut genomic DNA solution, 
20 uE of 10X T4 ligase buffer and 0.5 uE (200 U) of T4 ligase (see Note 5). 

7. Incubate at 16 C C for 12 h (overnight). 

8. Precipitate the circularized template DNA with ethanol, wash with 70% ethanol, and 
suspend DNA in 20 uE of sterile H 2 0. 

3.2. Inverse PCR Reaction 

1. In a PCR tube, combine the following components for the inverse PCR amplification 
reaction: 10 uE of 10X PCR buffer, 6 uE of 25 mM MgCl 2 (final concentration of 1 .5 mM), 
2 uE 10 mM dNTP mix (final concentration of 200 \iM each dNTP), 0.5 \iM of each 
primer, 10 uE (500 ng) of circularized template and sterile redistilled H 2 to 99.25 uE. 
Mix the components and centrifuge briefly in a microfuge. 

2. In a thermocycler, heat the reaction mix to 95°C for two minutes before adding 0.75 uE 
(2.6 U) of the polymerase enzyme mix ("hot start" PCR is used to improve the specificity, 
sensitivity, and yield of the reaction). 

3. Amplify the target DNA sequence using the following cycle profile: first five cycles: 
94°C for 30 s (denaturation), 60°C for 30 s (annealing) and 72°C (< 3kb) or 68°C (>3 kb) 
for 1 min/1 .5 kb (elongation). For the last 25-30 cycles reduce the annealing temperature 
to 55 °C. 

4. Analyze samples on a 0.7% agarose gel. 

3.3. Alternative Method 

Huang et al. (14) described the use of a nested PCR primer from the IS50 regions to 
distinguish the target product from nonspecific IPCR products. Using a nested primer 
set and two PCRs, it is possible to forgo the Southern blot analysis to determine the 
size of the PCR product (see Fig. 1). 

1. Prepare the IPCR template as described in the materials and methods. 

2. Set up a pair of PCR reactions for each template. Primer pairs to amplify either both, the 
left or right side of Tn5 are listed in Table 2. 

3. The extension step of the temperature cycle profile should be lengthened to 10 min to 
ensure that long target sequences will be amplified. 

4. Analyze the PCR products on a 0.7% agarose gel. Bands of the same size in both PCR and 
nested PCR indicate which is the correct product for the target sequence. 

3.4. Cloning and Sequencing of IPCR Products 

Because of the nature of the inverted terminal repeats of Tn5, it does not contain 
unique sites for sequencing primers to anneal at its ends. Therefore, IPCR products from 
templates generated by enzymes not cutting the transposon must be cloned first in order 
to provide priming sites for sequencing. This is achieved by blunt-end or TA cloning. 



Table 2 

List of Primer Pairs for Nested Tn5 IPCR 



CO 

IV) 



Enzyme used in 














template preparation 




Xmal 






Sail 




Side of Tn5 flanking 














DNA amplified 


Both 


Left 


Right 


Left 




Right 


Primer pair 


IRl fe 


UTn5< 


UTn5'' 


UTn5< 




UTn5'' 






XmaITn5L 


XmaITn5R 


SalITn5L 




SalITn5R 


Nested primer pair 


UTn5* 


XmaITn5U 


XmaITn5U 


SallTnSU 




SalITn5U 






XmaITn5L 


XmaITn5R 


SalITn5L 




SalITn5R 



Sphl 



BamUl 



Left 



Right 



Left 



Right 



UTW UTn5 r UTn5< UTn5 c 

SphTn5L SphTn5R BL BR 

SphITn5U SphITn5U IR2 IR2 

SphTn5L SphTn5R BL BR 



"The PCR product from these reactions can be cloned with cohesive ends introduced with the nested primer. 
h A single oligonucleotide primer is used for these PCRs. 
'This primer can be substituted with IR1, if desired. 



322 Martin and Mohn 

However, sequencing of the IPCR products without cloning is possible from 
amplicons of the left or right sides of Tn5 using the UR1 or UTn5 primers. 
Furthermore, the addition of a restriction endonuclease recognition site in the nested 
primers makes possible cohesive-end cloning of the PCR product (see Fig. 1) 

4. Notes 

1. * These enzymes can be blocked by overlapping DNA methylation sites. ** These 
enzymes leave a blunt-end on the cut DNA and may require the addition of PEG (16) or 
increased enzyme concentration in the reaction to increase ligation efficiency. 

2. To simplify the preparation of the DNA probe, sub-clone the Hindlll-BamHl fragment 
into a high-copy vector such as pUC19 and select on LB-kanamycin. Sufficient amounts 
of probe can be made by PCR using the Ml 3 primers or by isolating the fragment from 
the high copy vector. 

3. Finding the proper condition for washing the membrane is important because the asym- 
metric DNA probe partially hybridizes to the right IS50 target sequence and is easily 
washed off if the conditions are too stringent. Slowly increasing the wash stringency and 
using a phosphorimager with 1 h exposures to look at the blot between washes will ensure 
that you do not overwash the blot. 

4. Using Taq DNA polymerase, the maximum size of the IPCR products we were able to 
amplify from a genomic template was approx 3 kb. The length of the IPCR product 
amplified may be increased by using a proofreading polymerase or a blend of the two 
enzymes. Huang et al. (14) were able to amplify, at a low yield, a 6.2 kb IPCR product 
using a Taq/GB-D polymerase enzyme blend. However, an additional limitation to the 
size of the IPCR fragment amplified is the low probability of intramolecular ligation of 
long DNA fragments. 

5. Ligation reactions must be performed with dilute DNA samples (<5u.g/mL) to favor intra- 
molecular ligation. 

References 

1. Goryshin, I. Y., Miller, J. A., Kil, Y. V., Lanzov, V. A., and Reznikoff, W. S. (1998) Tn5/ 
IS50 target recognition. Proc. Natl. Acad. Sci. USA 95, 10,716-10,721. 

2. Berg, D. E. (1989) Transposon Tn5 in Mobile DNA, (Berg, D. E. and Howe, M. M., eds.), 
American Society for Microbiology, WA, pp. 163-184. 

3. De Lorenzo, V., Herrero, M., Jakubzik, U., and Timmis, K. N. (1990) Mini-Tn5 transposon 
derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of 
cloned DNA in gram-negative eubacteria. J. Bacteriol. 172, 6568-6572. 

4. Simon, R., Ouandt, J., and Klipp, W. (1989) New derivatives of transposon Tn5 suitable 
for mobilization of replicons, generation of operon fusions and induction of genes in 
Gram-negative bacteria. Gene 80, 161-170. 

5. DeBruijn, F., J. and Rossbach, S. (1993) Transposon mutagenesis in Methods for General 
and Molecular Bacteriology, (Gerhardt, P., ed.), American Society for Microbiology, WA, 
pp. 387-405. 

6. Dennis, J. J. and Zylstra, G. J. (1998) Plasposons: Modular self-cloning minitransposon 
derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl. Environ. 
Microbiol. 64,2710-2715. 

7. Hozbor, D. F., Pich Otero, A. J. L., Wynne, M. E., Petruccelli, S., and Lagares, A. (1998) 
Recovery of Tn5-flanking bacterial DNA by vector-mediated walking from the transposon 
to the host genome. Analyt. Biochem. 259, 286-288. 



Isolation of Tn5 Flanking DNA by IPCR 323 

8. Ribot, E. M., Quinn, F. D., Bai, X., and Murtagh, J. J. (1998) Rapid amplification of 
transposon ends for the isolation, cloning and sequencing of transposon-disrupted 
chromosomal genes. Biotechniques 24, 16-22. 

9. Prod'hom, G., Lagier, B., Pelicic, V., Hance, A. J., Gicquel, B., and Guilhot, C. (1998) A 
reliable amplification technique for the characterization of genomic DNA sequences 
flanking insertion sequences. FEMS Microbiol. Lett. 158, 75-81. 

10. Karlyshev, A. V., Pallen, M. J., and Wren, B. W. (2000) Single-primer PCR procedure for 
rapid identification of transposon insertion sites. Biotechniques 28, 1078-1081. 

11. Subramanian, P. S., Versalovic, J., McCabe, E. R., and Lupski, J. R. (1992) Rapid 
mapping of Escherichia coli:Tn5 insertion mutations by REP-Tn5 PCR. PCR Meth. 
Appl. 1, 187-192. 

12. Rich, J. J. and Willis, D. K. (1990) A single oligonucleotide can be used to rapidly isolate 
DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction. 
Nucl. Acid Res. 18, 6673-6676. 

13. Martin, V. J. J. and Mohn, W. W. (1999) An alternative inverse PCR (IPCR) method to 
amplify DNA sequences flanking Tn5 transposon insertions. ./. Microbiol. Meth. 35, 
163-166. 

14. Huang, G., Zhang, L., and Birch, R. G. (2000) Rapid amplification and cloning of Tn5 
flanking fragments by inverse PCR. Lett. Appl. Microbiol. 31, 149-153. 

15. Sambrook, J. and Russell, D. W. (2001) Molecular cloning: A laboratory manual. Cold 
Spring Harbor Laboratory Press. 

16. Upcroft, P. and Healey, A. (1987) Rapid and efficient method for cloning of blunt-ended 
DNA fragments. Gene 51, 69-76. 

17. Mitchell, D. andSmit, J. (1990) Identification of genes affecting production of the adhesion 
organelle of Caulobacter crescentus CB2. ./. Bacteriol. 172, 5425-5431. 



34 



Rapid Amplification of Genomic DNA Sequences 
Tagged by Insertional Mutagenesis 

Martina Celerin and Kristin T. Chun 
1. Introduction 

Current and recent efforts to determine the genomic DNA sequence for numerous 
organisms (e.g., Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, 
Arabidopsis thaliana, Zea mays, Caenorhabditis elegans, Mus musculus, Homo sapi- 
ens, Schizosaccharomyces pombe, Danio rerio, Drosophila melanogaster, Oryza 
sativa, and various archaea, and eubacteria) have revealed novel genes with unknown 
functions, and transposon mutagenesis provides a powerful method for assigning func- 
tions to these genes (e.g., 1,2-5). In addition, restriction enzyme-mediated integration 
(REMI) has been used widely for mutagenesis (e.g., 6,7, reviewed in 8). For both of 
these approaches, a unique DNA sequence is inserted by nonhomologous integration 
into genomic DNA to generate a disruption mutation that is physically tagged. Genetic 
analysis of the resulting mutants yields those with defects in the function of interest. 
Because each mutation is physically marked by a unique DNA sequence, it is possible 
to use this tag to identify each disrupted gene. 

The large-scale characterization of these mutations requires a reliable, rapid, and 
simple method to identify the site of insertion. The polymerase chain reaction (PCR) 
enables the quick and easy identification and analysis of specific DNA sequences, but 
in its original form, this process requires knowledge of the DNA sequences at each 
end of the DNA being amplified (9). For collections of transposon or REMI-generated 
disruption mutations, the DNA sequence of the inserted DNA is known, but the 
sequence of the surrounding gene is not. Involving only two successive PCRs and two 
pairs of PCR primers, semi-random, two-step PCR (ST-PCR) provides a simple, rapid 
method for identifying these disrupted genes. 

ST-PCR for disruption mutations in S. cerevisiae consists of two successive PCRs 
utilizing two different pairs of PCR primers. When used to amplify disruptions from 
Coprinus cinereus (whose genome is roughly three times larger than that of S. 
cerevisisae), a third PCR is sometimes used. For the first reaction, the template is 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

325 



326 Celerin and Chun 

primer 3 primer 1 

^ chromosomal DNA mmm ^^^^^^^^^ 

irimer 2 

transposon 




5' GGCCACGCGTCGACTAGTAC-NNNNNNNNNN-GAIAI 3' 
defined sequence <*%££ 

Fig. 1. Summary of ST-PCR scheme. (Reproduced from ref. 10 with permission) 



genomic DNA from the mutant of interest, and primers 1 and 2 direct amplification 
{see Fig. 1). Primer 1 anneals specifically near one known end of the inserted DNA 
(e.g., transposon or REMI DNA element) and directs DNA polymerization away from 
that end and into the disrupted genomic DNA. Primer 2 contains a specific 20-nucle- 
otide sequence followed by 10 bases of degenerate sequence and a specific five-nucle- 
otide sequence {see Fig. 1). The 20 defined bases provide the sequence to which a 
primer in the second PCR will anneal. For the 10 random bases, during the synthesis 
of primer 2, a mixture of all four nucleosides is incorporated at each of the degenerate 
base positions, so in this region, the resulting collection of primers should include 
every possible permutation. The specific five-nucleotide sequence at the 3' end is pre- 
dicted to occur every 500 to 1500 bp of the genome being analyzed. In the S. cerevisiae 
genome, which consists of approx 40% G+C, the sequence GATAT should occur 
approx every 600 bp. Assuming the occurrence of this sequence follows a Poisson 
distribution, there is an 80% chance that at least one "GATAT" occurs within 1 kb of 
any position in the genome and a 90% chance that one occurs within 1.5 kb {see 
Fig. 2). Therefore, at least one "GATAT" should be found near each transposon 
insertion. Consequently, at least one of the primer 2's is likely to anneal to a GATAT 
site near the insertion and, along with primer 1, promote the amplification of the 
DNA sequence in and next to the mutation. 

The annealing temperatures for the first six rounds of the first amplification are rela- 
tively low to allow primer 2's with low melting temperatures as well as those with some 
mismatches to anneal {see Table 1). If the 15 3'-most bases of primer 2 contribute the 
most to its melting temperature {T m ), a primer with 10 A's and/or T's in the degen- 
erate region will have a predicted 7m of about 32°C. Alternatively, a primer with 10 G's 
and/or C's will have a T m of about 52°C. To favor primers that anneal under relatively 
stringent conditions, the first annealing temperature is 42°C, and for each of the next five 
PCR cycles, the annealing temperature is one degree cooler (41°C and so on). 

For the second PCR, PCR products from the first reaction serve as the template, 
and a second pair of primers direct amplification. Primer 3 anneals to the inserted 
DNA element, nested relative to primer 1 , and primer 4 anneals to the complement of 



Mutant Analysis by ST-PCR 



327 



1.00 



0.80 



— 0.60 



n 

(0 

.n 
o 



0.40- 



0.20 



0.00 




0.0 



0.5 



1.0 



1.5 



2.0 



2.5 



Interval (kb) 



Fig. 2. Probability that the sequence "GATAT" will occur within a given interval of S. 
cerevisiae genomic DNA. This graph assumes that the occurrence of "GATAT" occurs 
randomly and conforms to a Poisson distribution and that the 5. cerevisiae genome consists of 
40% G+C basepairs. 



Table 1 

DNA Primers Used in ST-PCR 



Primer 

1 

2/OL-106 

3 

4/OL-107 

OL-103 

OL-104 

OL-105 

OL-108 

OL-109 

OL-110 



DNA sequence (5' to 3') 

TAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGA 

GGCCACGCGTCGACTAGTAC (N) 10 GATAT 

(CAU) 4 TGATAATCTCATGACCAAAATCCC 

(CUA) 4 GGCCACGCGTCGACTAGTAC 

(CAU) 4 GTCGCGGTGAGTTCAGGCTT 

ATCCACGCCCTCCTACATCG 

(CAU) 4 CAGCTTCATTTTCCGTGTGG 

(CUA) 4 TAGAATACTCAAGCTATGCA 

CCGGTGCGCAGCTGATCATG(N 10 )CTATA 

(CAU) 4 CCGGTGCGCAGCTGATCATG 



328 Celerin and Chun 

the 20 bases of defined sequence at the 5' end of primer 2 (see Fig. 1). These primers 
are intended to amplify the PCR products from the first reaction that were specifically 
generated from primers 1 and 2, and not to amplify PCR products generated from pairs 
of primer 2's. The resulting PCR product can be used as the template for direct DNA 
sequence analysis to determine the sequence of the disrupted genomic DNA. Using 
this method, we rapidly and easily identified the essential genes in a collection of 
mini-Tn3 transposon mutated S. cerevisiae mutants (2,10,11). 

With a few modifications, we also used this method to identify the mutated genes in 
a collection of C. cinereus sporulation-defective mutants (7). These mutations were 
generated by REMI, whereby C. cinereus was cotransformed with a restriction endo- 
nuclease and a linearized plasmid, pPHTl (7 and Fig. 3). This plasmid contains PHT1, 
a dominant selectable marker which consists of the open reading frame of the E. coli 
hygromycin B phosphotransferase gene (hph, 12) fused to the promoter and termina- 
tor regions of the C. cinereus (3-tubulin gene. This cotransformation results in the 
reasonably random, nonhomologous integration of the selectable marker and the gen- 
eration of a disruption mutation at the insertion site (6,7, reviewed in 8). We deter- 
mined that during REMI mutagenesis of C. cinereus, roughly 50% of the insertion 
events are nonperfect integrations (7). These are likely to result from degradation of 
the ends of the transforming DNA and processing of the restriction enzyme-generated 
double-strand breaks in the target DNA prior to insertion, as well as integration of the 
transforming DNA independent of the restriction enzyme cleavage site. If substantial 
truncation of the transforming DNA occurs, terminal-proximal priming sites may be 
lost. Therefore, unlike ST-PCR in S. cerevisiae for transposon-disruption mutants for 
which only two pairs of primers were used, several different combinations of primers 
were designed to amplify REMI-generated disruptions for C. cinereus. By using this 
approach, we were able to analyze mutants in which the pPHTl was truncated during 
integration. Three different primer pairs anneal to pPHTl, either near its hph end or to 
a proximal region (e.g., to the 6-tubulin promoter or to hph). In addition, a fourth pair 
of primers was designed to anneal to the opposite end of pPHT 1 . 

Numerous other modifications of PCR that allow the amplification of unknown 
nucleotide sequences next to known sequences have been described. The concept of 
using a known cDNA sequence and its 3' polyA tail to amplify the intervening 
sequence was introduced with RACE (13), one-sided PCR (14), and anchored PCR 
(15). When seeking DNA sequences that are not at the 3' end of a cDNA (and lack the 
poly A tail), these procedures all require terminal deoxy nucleotidyl-transferase to 
attach to the end of the unknown sequence a polynucleotide tail. Inverse PCR (16), 
inverted PCR (17), ligation-mediated single-sided PCR (18), single-specific primer 
PCR (SSP-PCR) (19), and panhandle PCR (20), all require the generation of a DNA 
fragment containing the known DNA sequence as well as the adjacent unknown 
sequences of interest (for example, restriction enzyme digestion). In addition, these 
five methods require the ligation of a known DNA sequence to the unknown end. 
Rapid amplification of transposon ends (RATE), ligation-mediated PCR (LMPCR), 
and vectorette-mediated PCR also require a ligation step (21-23). Transposon inser- 



Mutant Analysis by ST-PCR 



329 



I "SI'S 




genomic DNA (Coprinus cinereus) 

pPCRII 

beta-tubulin promotor/terminator 

hph 



Primer set 
A 
B 
C 
D 



PCR1 
OL-104/OL-106 
OL-103/OL-106 
OL-104/OL-106 
OL-111/OL-109 



PCR2 
OL-105/OL-107 
OL-105/OL-107 
OL-103/OL-107 
OL-108/OL-110 




MDL111 
-OL108 

OL110 
OL109 



Fig. 3 . ST-PCR strategy used for cloning the genomic DNA sequences adjacent to the pPHT 1 
insert in C. cinereus mutants. 



tion display (TID) requires both restriction endonuclease digestion and ligation (24). 
In these types of modified PCR, one primer anneals to the DNA of known sequence, a 
second anneals to the sequences added to the other end, and the intervening, unknown 
sequences are amplified. 

Several other PCR protocols (e.g., targeted gene walking, 25) employ a primer that 
anneals specifically at or near the end of a region of known sequence and a collection 
of primers that anneal imperfectly to a nearby, unknown sequence. Nonstringent 
annealing conditions allow the second primers to anneal. A similar method, PCR medi- 
ated by a single primer (26), uses only one primer and relies on conditions where it 
anneals specifically to its complementary sequence as well as imperfectly to a nearby 
sequence. These methods require none of the enzymatic steps described earlier, but 
they often yield multiple PCR products, because in addition to the expected DNA 
fragment, they yield products generated from two primers annealing imperfectly to 
genomic target sequences. The multiple PCR products generated must be screened by 
Southern analysis to identify the desired fragment. 

Finally, thermal asymmetric interlaced (TAIL-) PCR has been used to amplify the 
genomic sequences flanking insertions in A. thaliana (27) and C. cinereus (P. Medina 
and P. Pukkila, personal communication). Like ST-PCR, it does not require additional 
steps before PCR, and it does not require screening afterward. This method involves 
three successive PCRs. 



330 Celerin and Chun 

ST-PCR is a simple, efficient, and rapid method to identify novel DNA sequences 
next to previously known sequences. In addition to analyzing single sequences of 
interest, it should also be applicable to large-scale analyses. Although we have used 
this method to analyze mutations in fungi, it should be possible to modify it for exam- 
ining more complex genomes. 

2. Materials 

1. Thermal cycler (PCR) machine. Ideally, it should have a program function that allows a 
progressive decrease of temperature each time a step in a cycle subroutine is executed. 

2. Taq polymerase. We successfully used enzyme from Roche (Indianapolis, IN) for ST- 
PCR from S. cerevisiae and from Fisher Scientific (Pittsburgh, PA) for amplification 
from C. cinereus. It is likely that enzyme from other sources will also be suitable. 

3. PCR buffer. 10X stock (500 mM KC1, 100 mM Tris-HCl pH 8.3, 2 mg/mL gelatin, 30 mM 
MgCl 2 , and 2 mM of each dNTP) was used for Taq from Roche. The 10X buffer supplied 
by Fisher Scientific was used for Taq from Fisher Scientific, and this reaction was supple- 
mented with MgCl 2 to a final concentration of 4.5 mM. All reagents should be molecular 
biology grade. Solutions should be made with double-distilled or ultrapure water and 
should be autoclave sterilized (except the TAE or TBE buffer for agarose gel elector- 
phoresis, which does not need to be sterile). 

4. PCR primers. The sequences for the PCR primers we use are listed in Table 1 and shown 
in Figs. 1 and 3. One set of primers was designed to amplify mini-Tn3 transposon dis- 
rupted sequences in S. cerevisiae and another set was for amplifying REMI-generated 
pPHTl -disrupted sequences in C. cinereus. Therefore, for other applications, four or more 
primers should be designed to suit the characteristics of the inserted DNA and the genome 
being studied. 

Primer 1 should anneal near one end of the inserted DNA and direct DNA polymeriza- 
tion through that end and into the disrupted genomic DNA. 

Primer 2 consists of 20 bases of defined sequence, followed by 10 random bases, 
followed by five defined bases. The 20 defined bases provide the sequence to which a 
primer in the second PCR will anneal. For S. cerevisisae, we chose the 20 base sequence 
used in the 5' RACE primers sold by Life Technologies, Gibco-BRL, but any other unique 
sequence should work well. For the ten random bases, during the synthesis of primer 2, a 
mixture of all four nucleosides is incorporated at each of the degenerate base positions. 
Alternatively, nucleosides containing 2'-deoxyinosine or a universal base, such as 
3-nitropyrrole or 5-nitroindole, could be used as degenerate bases (28-30). The 3'-most five 
base sequence should correspond to a sequence expected to occur in the genome close 
enough to the insertion site to allow efficient amplification of the intervening sequence. 
Consequently, assuming that the 15 3'-most bases of primer 2 are the most important for 
annealing to the template, at least one of these primers should be likely to anneal to the 
complement of the five base sequence occurring near the insertion site and, along with 
primer 1, promote the amplification of the DNA sequence in and next to the mutation. 

Primer 3 anneals to the inserted DNA element (in our case mini-Tn3 orpPHTl), nested 
with respect to primer 1, and primer 4 anneals to the complement of the 20 bases of 
defined sequence at the 5' end of primer 2. These primers are intended to amplify the PCR 
products from the first reaction that were specifically generated from primers 1 and 2. 

5. To analyze PCR products, standard agarose gel electrophoresis equipment and molecular 
biology grade agarose is used. To purify PCR products after agarose gel electrophoresis, 
standard methods [e.g. Geneclean (Bio 101) or QiaexII (Qiagen) kits] work well. 



Mutant Analysis by ST-PCR 33 1 

6. 50X TAE or 10X TBE is used as the stock for agarose gels and running buffer. 50X TAE: 
2.0 M Tris-acetate, 50 mM EDTA, adjust pH to 7.9 with glacial acetic acid. 10X TBE: 0.9 M 
Tris-borate, and 20 mM EDTA adjusted pH to 8.0 with NaOH. 

7. To determine the DNA sequence of the amplified DNA, the PCR product may be 
sequenced directly using a thermal cycle sequencing protocol. Alternatively, if primer 3 
is designed to contain (CAU) 4 at its 5' end and primer 4 contains (CUA) 4 at its 5' end (in 
addition to the sequences for these primers described earlier), the Clone-Amp pAMPl 
System for Rapid Cloning of Amplification Products (Life Technologies, Gibco-BRL) 
may be used to subclone the PCR fragment directionally into the pAMPl plasmid, and 
the resulting recombinant can then be sequenced using standard methods. This DNA 
sequence can then be compared to the complete sequence of the S. cerevisiae genome to 
identify the complete sequence of the disrupted gene. 

8. TE buffer: 10 mM Tris-HCl pH 7.4-8.0, 1.0 mM EDTA. 

3. Method 

3.1. ST-PCR from Mini-Tn3 Disruption Mutations in S. cerevisiae 

1. Purify genomic DNA containing the disruption mutation using standard methods. Any 
method that yields DNA of sufficient purity for restriction endonuclease digestion or 
genomic blot analysis should work well. Resuspend the DNA in TE buffer or ultrapure 
water. 

2. For the first PCR (PCR1, 20 uL total volume) mix approximately 100 ng of template 
DNA, IX PCR buffer, 20 pmol each of PCR primers 1 and 2, and 2.5 units of Taq poly- 
merase. Reagents and each reaction should be stored on ice during preparation. Carry out 
this first amplification reaction using the following program: 

a. 94°C, 2min 

b. 94°C, 30 s 

c. Initial temp. 42°C, 30 s. Decrease 1.0°C for each subsequent cycle 

d. 72°C, 3min 

e. Return to step b and repeat five more times 

f. 94°C, 30 s 

g. 65°C, 30 s 
h. 72°C, 3min 

i. return to step f and repeat this subroutine 24 more times 
j. 4°C, hold 
k. End 

3. Dilute the resulting PCR with 80 |xL of sterile water, and use 1 uL of this as the template 
for the second PCR (PCR2), which is essentially the same as the first, except primers 3 
and 4 and the following PCR program are used: 

a. 94°C, 30 s 

b. 65°C, 30 s 

c. 72°C, 3min 

d. return to step a and repeat this subroutine 29 more times 

e. 4°C, hold 

f. End 

4. Load 5 \iL of the 20 |xL reaction onto a 1 .0% agarose gel in 0.5X TAE or 1 .OX TBE and 
separate the PCR products using a standard electrophoresis gel box and power supply. 
0.5 fxg/mL ethidium bromide may be included in the gel or the gel may be stained after 
electrophoresis. 



332 Celerin and Chun 

5. Excise the PCR fragment from the gel using a razor blade or scalpel and purify the DNA 
from the agarose using standard methods (e.g., Geneclean (BiolOl) or QiaexII (Qiagen) 
kit) into 20 uE of TE buffer. 

6. The DNA sequence of the purified fragment can then be determined directly using a 
thermal cycle sequencing protocol, 5 uE of the 20 uE of PCR2 fragment, and primer 3 as 
the sequencing primer. Alternatively, one can subclone the PCR2 fragment into a plasmid 
vector. One approach to accomplish this is to use the Clone-Amp pAMPl System for 
Rapid Cloning of Amplification Products (Gibco-BRL). To do so, one would design 
primer 3 with (CAU) 4 at its 5' end and primer 4 with (CUA) 4 , at its 5' end. PCR2 frag- 
ments amplified with these primers will contain four uridines at the 5' end of each strand. 
Treatment of this DNA with uracil DNA glycosylase removes uracil, disrupts base pair- 
ing, and exposes the complementary single-strand 3' ends. These ends can then anneal to 
complementary single-strand pAMPl vector ends. Ligation is not required, so the 
annealed fragments can be transformed directly into competent E. coli. The resulting 
recombinant can then be sequenced using standard methods (see Note 1). 

3.2. ST-PCR from REMI-Generated Disruption Mutations 
in C. cinereus 

The ST-PCR protocol can be modified to amplify disrupted genomic sequences 
from genomes more complex than that of S. cerevisiae. Here, we describe modifica- 
tions used to analyze REMI-generated mutations in C. cinereus, whose genome is 
37.5 Mb, roughly three times larger than that of S. cerevisiae. 

1. Just as for S. cerevisiae, DNA from C. cinereus REMI-generated mutants that is suffi- 
cient for genomic blot analysis should work well for ST-PCR. 

2. The first PCR is the same as that described in Subheading 3.1., step 2, except for the 
following modifications. 

a. A set of primers (A, B, C, or D) is used instead of S. cerevisiae primers 1 and 2 (see 
Table 1 and Fig. 3). Primer sets A, B, and C were designed to amplify from the 
(5-tubulin promoter end of pPHTl ("head" end), and primer set D was designed to 
amplify form the other end ("tail" end) of pPHTl. Of course, additional primer sets 
could be designed (see Note 2). 

3. PCR1 is diluted as described in Subheading 3.1., step 3 for use as the template in the 
PCR2. The same PCR2 conditions are used as those described in Subheading 3.1., 
step 3, except for the following modifications. 

a. 5% (w/v) acetamide is added to the reaction (see Note 3). 

b. Only 20 cycles of amplification are used (see Note 3). 

c. Depending upon which primer pair was used in PCR1, the correponding pair for 
PCR2 is used in this subsequent PCR. (see Table 1, e.g., if OL-104 and OL-106 are 
used in PCR1, OL-105 and OL-107 are used in PCR2) 

4. PCR2 fragments are resolved by agarose gel electrophoresis (see Note 4) and purified as 
described in Subheading 3.1., steps 4 and 5. 

5. Several of the C. cinereus PCR2 amplifications generated only a small quantity of prod- 
uct. To increase the likelihood of successfully cloning these products, a third cycle of 
amplification can be used. In this third PCR, 1 uE of the 20-uE eluate from the excised 
PCR2 fragment is used as the template, and otherwise the reaction mix and conditions are 
as described for the PCR2 (see step 3 above). The products of the amplification are sepa- 
rated by agarose gel electrophoresis, and the DNA fragments are excised, cloned into 
pAMPl (as described above for S. cerevisiae), and sequenced (see Notes 5 and 6). 



Mutant Analysis by ST-PCR 333 

6. For PCR2 amplifications involving primer sets A, B, or C (see Fig. 3), the resulting prod- 
ucts include a portion of the C. cinereus (3-tubulin promoter from pPHTl. Each product 
from these amplifications should be screened by Southern analysis, and only those with 
this expected portion of pPHTl should be analyzed further (see Note 5). 

7. Depending upon the primer set used, each of the ST-PCR products should contain a pre- 
dicted portion of amplified pPHTl. For ST-PCR amplifications involving primer sets A 
or B (see Fig. 3), the product should contain 181 bp of pPHTl. Ideally, the size of the 
genomic DNA amplified should be greater than 0.3 kb, but 100 bp can be sufficient and 
therefore any ST-PCR products greater than 0.3 kb are pursued. For similar reasons, only 
products greater than 0.6 kb and 0.2 kb from ST-PCR amplifications involving primer 
sets C or D, respectively, are pursued (see Fig. 3, Note 4). 

8. The DNA fragment corresponding to the region of disrupted genomic DNA can be used 
to probe a blot of separated chromosomes to map the mutated gene to a chromosome. 
This probe can subsequently be used to probe either a corresponding chromosome-spe- 
cific or a genomic cosmid library to isolate a complete genomic clone. 

4. Notes 

1. In theory, PCR2 might generate, in addition to a single PCR product amplified with prim- 
ers 3 and 4, one or more PCR products amplified with a pair of primer 4's (that had 
annealed to a product from the first PCR generated from two primer 2's). Surprisingly, 
we did not observe such PCR products. It is possible that this is not a common occurrence 
and would only be observed after analysis of many more mutants. However, if the prod- 
ucts from the second PCR are cloned into a vector in a way that requires that the ends of 
the insert contain primers 3 and 4, these extraneous PCR products are eliminated. In our 
case, specific DNA sequences were incorporated into the 5' ends of primers 3 and 4, and 
these were used to insert the PCR product asymmetrically into a compatible plasmid (see 
Subheadings 2.7. and 3.1.6.). 

2. It is useful to know the number of PHT1 integration sites in the genome of the REMI 
transformant being analyzed. We determined this by probing with hph Southern blots of 
restriction-digested genomic DNA and intact, resolved chromosomes. REMI 
transformants with multiple sites of insertion were backcrossed to obtain isolates that 
contain a single site of integration. Also, depending on the relative orientation of multiple 
inserts at a single locus, some primer pairs were not useful. For example, in a tail-to-tail 
orientation, primer combination D (see Fig. 3) would not be expected to amplify flanking 
genomic DNA, whereas primer combinations A, B, or C might. Therefore, to predict 
which primer pairs should be used, the number of pPHTl constructs per site of integra- 
tion, as well as their relative orientation should be determined. We used restriction digests 
and Southern blot analysis to determine the orientation of inserts within a site (e.g., head- 
to-tail, head-to-head, tail-to-tail). 

3. We found that including 5% acetamide in the PCR2, as well as decreasing the number of 
amplification cycles to only 20 (instead of the 30 used for S.cerevisiae) decreased the 
amount of smearing when these products were analyzed on agarose gels. This, in turn, 
allowed us to see less abundant PCR products, which could be excised, purified, and used 
in a third PCR. We also tried using less of the first PCR as the template for the second 
PCR (instead of only 1:5, diluting the first PCR 1:10, 1:100, or 1:1000, before taking 
1 u,L for the second PCR). This modification decreased smearing, but unfortunately it 
also decreased product yield. 

4. For C. cinereus REMI-generated mutants, the ST-PCR products obtained ranged in size 
from 0.3 to 1.2 kb. 



334 Celerin and Chun 

5. Some of the products obtained in amplifications using primer sets A, B, or C did not 
contain the expected portion of the |5-tubulin promoter from pPHTl. The specific cause 
of these PCR artifacts is unknown, but these were observed infrequently. 

6. We found that in some of the PCR2 and PCR3 amplifications multiple products were 
generated. By cloning and sequencing these products, we determined that many were 
simply shorter or longer amplifications of the same genomic DN A. Additionally, some of 
the products turned out to be amplifications of the native (3-tubulin gene and were identi- 
fied as such during the sequencing step. 

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(1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. 
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10. Chun, K. T., Edenberg, H. J., Kelley, M. R., and Goebl, M. G. (1997) Rapid amplifica- 
tion of uncharacterized transposon-tagged DNA sequences from genomic DNA. Yeast 
13, 233-240. 

11. Chun, K. T. and Goebl, M. G. (1997) Mutational analysis of Caklp, an essential protein 
kinase that regulates cell cycle progression. Mol. Gen. Genet. 256, 365-375. 

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hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccha- 
romyces cerevisiae. Gene 25, 179-188. 

13. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full-length 
cDNAs from rare transcripts: amplification using a single gene-specific olignucleotide 
primer. Proc. Natl. Acad. Sci. USA 85, 8998-9002. 

14. Ohara, O., Dorit, R. L., and Gilbert, W. (1989) One-sided polymerase chain reaction: the 
amplification of cDNA. Proc. Natl. Acad. Sci. USA 86, 5673-5677. 



Mutant Analysis by ST-PCR 335 

15. Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., and Davis, M. M. (1989) Polymerase 
chain reaction with single-sided specificity: analysis of T cell receptor delta chain. Science 
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16. Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse 
polymerase chain reaction. Genetics 120, 621-623. 

17. Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) A procedure for in vitro amplifica- 
tion of DNA segments that lie outside the boundaries of known sequences. Nucl. Acids 
Res. 16,8186. 

18. Mueller, P. R. and Wold, B. (1989) In vivo footprinting of a muscle specific enhancer by 
ligation mediated PCR. Science 246, 780-786. 

19. Shyamala, V. and Ames, G. F. (1989) Genome walking by single-specific-primer 
polymerase chain reaction: SSP-PCR. Gene 84, 1-8. 

20. Jones, D. H. and Winistorfer, S. C. (1992) Sequence specific generation of a DNA panhan- 
dle permits PCR amplification of unknown flanking DNA. Nucl. Acids Res. 20, 595-600. 

21. Ribot, E. M. (1998) Rapid amplification of transposon ends for the isolation, cloning and 
sequencing of transposon-disrupted chromosomal genes. Biotechniqu.es 24, 16-22. 

22. Prod'hom, G., Lagier, B., Pelicic, V., Hance, A. J., Gicquel, B., and Guilhot, C. (1998) A 
reliable amplification technique for the characterization of genomic DNA sequences flank- 
ing insertion sequences. FEMS Microbiol. Let. 158, 75-81. 

23. Eggert, H., Bergemann, K., and Saumweber, H. (1998) Molecular screening of P-element 
insertions in a large genomic region of Drosophila melanogaster using polymerase chain 
reaction mediated by the Vectorette. Genetics 149, 1427-1434. 

24. Yephremov, A. and Saedler, H. (2000) Display and isolation of transposon-flanking 
sequences starting from genomic DNA and RNA. Plant J . 21, 495-505. 

25. Parker, J. D., Rabinovitch, P. S., and Burmer, G. C. (1991) Targeted gene walking 
polymerase chain reaction. Nucl. Acids Res. 19, 3055-3060. 

26. Parks, C. L., Chang, L. S., and Shenk, T. (1991) A polymerase chain reaction mediated by 
a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein 
coding region. Nucl. Acids Res. 19, 7155-7160. 

27. Liu, Y.-G., Mitsukawa, M„ Oosumi, T., and Whittier, R. F. (1995) Efficient isolation and 
mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric 
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28. Knoth, K., Roberds, S., Poteet, C, and Tamkun, M. (1988) Highly degenerate, inosine- 
containing primers specifically amplify rare cDNA using the polymerase chain reaction. 
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29. Nichols, R., Andrews, P. C, Th&ng, P., and Bergstrom, D. E. (1994) A universal nucleo- 
side for use at ambiguous sites in DNA primers. Nature 369, 492-493. 

30. Loakes, D., Brown, D. M., Linde, S., and Hill, F. (1995) 3-Nitropyrrole and 5-nitroindole as 
universal bases in primers for DNA sequencing and PCR. Nucl. Acids Res. 23, 2361-2366. 



35. 



Isolation of Large-Terminal Sequences of BAC Inserts 
Based on Double-Restriction-Enzyme Digestion 
Followed by Anchored PCR 

Zhong-Nan Yang and T. Erik Mirkov 
1. Introduction 

Large insert libraries are critical in genome-related research. Bacterial artificial 
chromosome (BAC) libraries are widely used in plant, animal, and human research; 
the ends of BAC clones are used as probes for chromosome walking and to confirm 
overlapping of contigs, as well as RFLP markers for mapping. Several methods have 
been developed to isolate BAC ends, including subcloning methods, plasmid rescue, 
and polymerase chain reaction (PCR)-based methods such as inverse PCR, thermal 
asymmetric interlaced PCR (TAIL-PCR), and Vectorette PCR (1, and references 
therein). Here, we described BAC end isolation based on double-restriction-enzyme 
digestion followed by anchored PCR. The BAC vector in the examples used here is 
pBeloBACll (2). Other vectors can be used, but the specific designation of left or 
right ends as used here may be different depending on the particular vector used. 

The strategy to isolate BAC ends is outlined in Fig. 1. The first step is the double- 
restriction-enzyme digestion of the BAC DNA. The two enzymes used are Notl and 
any one of the following four enzymes: EcoRV, Hpal, StuI, andXmnl. There are only 
two Notl sites in the BAC vector flanking the insert, and Notl would be expected to cut 
infrequently in the inserts. For the second enzyme of the double digestion, any enzyme 
with a 6-basepair recognition sequence that is not present in the left or right arms of 
the BAC vector can be used. Here the four enzymes EcoRV, Hpal, StuI, and Xmnl are 
chosen as they leave blunt termini after digestion. EcoRV is used as representative of 
the second enzyme in an example of the isolation strategy in Fig. 1. After double 
digestion, the DNA fragments containing the left arm (T7 side) with the left end of 
the BAC insert and the right arm (SP6 side) with the right end of the BAC insert will 
have one blunt termini and a Notl termini (L and R in Fig. 1). The second step is 
dephosphorylation of digested BAC DNA, which prevents the digested BAC DNA 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

337 



338 



Yang and Mirkov 



NotI K H 


E 


E 


E E 


E 
^1 


H NotI 
SP6 


-*• 
Fbac 






Double digestion 




<*- 
Rbac 


NotI T7 K H 


E 


E E E* E E E 


E 


H NotI 


(L)| | M 




Ml 


M W 




I SP6 1 

Ml 1 I™ 



Fbac 



Rbac 



E (Blunt) 




NotI K H E/E 

T7 



NotI 



H E/E 





Fig. 1. Schematic diagram of the strategy used for specific amplification of terminal 
sequences of BAC clones. B AC ends containing the left arm (L) or right arm (R) of the BAC 
vector will be released after double-restriction-enzyme digestion (NotI and £coRV). These 
DNA fragments are ligated to the NotI and EcoRV sites of pMSK. PCR products representing 
the left and right ends can be amplified specifically with primers Rsk and Fbac (left end) or Rsk 
and Rbac (right end). K, Kpnl; H, Hindlll; E, £coRV; Fbac, left arm specific primer; Rbac, 
right arm specific primer; Rsk, pMSK specific primer adjacent to the EcoRV site. T7, T7 
promoter; SP6, SP6 promoter. 



from being religated. The third step is the ligation of digested BAC DNA with the NotI 
and ficoRV-digested anchor vector pMSK. The fourth step is PCR amplification with 
one primer annealing to pMSK and another primer annealing to the left arm (Fbac) or 
right arm (Rbac) to specifically amplify the left end or right end correspondingly. 

With the optimized conditions described in this protocol, the PCR product is a single 
strong band that can be used directly to generate probes for hybridization. In most 
cases, PCR products that represent BAC ends can be obtained from two or three of the 
four double digestions and ligations. This allows reconfirmation of isolated BAC ends 
simply by restriction enzyme digestion (1). For each BAC clone, four double-restric- 



Isolation of BAC Ends 339 

tion enzyme digestions were used. The actual probability to obtain PCR products that 
represent both the left and right ends would be expected to be greater than 90%. This 
method is simple, fast and reliable and it has become a routine method for BAC end 
isolation in our laboratory. 

2. Materials 

1. BAC DNA prepared from a 5-mL overnight culture and resuspended in 35 uL H 2 (3). 

2. Plasmid pBluescript II SK(+) (Stratagene, LA Jolla, CA) that contains the restriction sites 
Notl and EcoRV. 

3. GENECLEAN II Kit from Bio 101, Inc., (Carlsbad, CA). 

4. Restriction enzymes (see Note 1): Notl, EcoRV, Hpdl, Kpril, Pvull, Sad, Stul, Xmnl 
(20 U/uL) and their appropriate reaction buffer from New England Biolabs (Beverly, MA). 

5. Shrimp alkaline phosphatase (Amersham Pharmacia Biotech, Piscataway, NJ). 

6. T4 DNA ligase and its reaction buffer (Life Technologies, Rockville, MD) 

7. Primers: Rsk (5'-TCGAGGTCGACGGTATCG-3'); Fbac (5'-AGTCGACCTGCAG- 
GCATG-3'); and Rbac (5'-CGCCAAGCTATTTAGGTGA-3'), each at 1 u.g/uL. 

8. 5 U/uL Taq DNA polymerase and 10X Taq reaction buffer. 

9. 2 mM deoxynucleotide 5'-triphosphate (dNTP) mix (final concentration of 2 mM of each 
of the four dNTPs). 

10. IX TAE buffer: 0.04 M Tris-acetate, 0.001 M ethylenediaminetetraacetic acid (EDTA), 
pH 8.0. 

11. Mineral oil. 

12. Thin-walled PCR tubes. 

13. Automated thermal cycler. 

3. Methods 

3.1. Preparation of pMSK from pBluescript II SK(+) (see Note 2) 

After digestion of pBluescript II SK(+) with Sad and Kpnl, a 101-bp fragment 
containing the multiple cloning site is released. This fragment is made blunt with T4 
DNA polymerase, gel purified, and ligated to the gel purified 2.5-kb Pvull fragment of 
pBluescript II SK(+) containing the fl(+) origin, ColEl origin, and the ampicillin- 
resistance gene, to obtain the anchor vector pMSK (7). 

3.2. Anchor Vector Preparation 

1. Plasmid pMSK is used as an anchor vector to ligate with digested BAC DNA. It is pre- 
pared from pBluescript II SK(+) as described above. 

2. Prepare a mix by adding the following to a tube on ice: 

a. 4^gpMSKDNA; 

b. 8 (xL NEB Reaction Buffer 3; 

c. 4uL(10U/uL)NofI 

d. 4 uL (20 U/uL) EcoRV 

e. Add H 2 to a final volume of 80 uL. 
Incubate at 37°C for 2 h. 

3. Electrophorese the digestion in a 1% agarose gel in IX TAE buffer. 

4. Excise the band of 2.6 kb and purify with the GenClean II kit following the manufacturer's 
instructions. Elute the DNA in a final volume of 80 uL. 



340 Yang and Mirkov 

3.3. BAC DNA Double Digestion and Dephosphorylation 

1. Prepare a master mixture by adding the following to a tube on ice: 

a. 8 uE 10X NEB Reaction Buffer 4; 

b. 4 uL Miniprep BAC DNA (see Note 3); 

c. 4 nL Notl (10 U/[aL); 

d. 60ixLH 2 O. 

Aliquot 19 (xL of the mixture into each of four tubes. Add 1 uE of one of the second 
restriction enzyme (EcoRW, Hpal, Stul, or Xmnl) to each tube and incubate at 37°C 
for 1 h. 

2. Add 0.5 [xL (0.5 U) shrimp alkaline phosphatase to each tube and incubate at 37°C for 30 min. 

3. Incubate at 65 C C for 20 min to inactivate the shrimp alkaline phosphatase. 

3.4. Ligation (PCR Template Preparation) 

1. Prepare a master mixture by adding the following to a tube on ice: 

a. 16 uE EcoRV and Notl digested pMSK vector; 

b. 16 [xL 5X T4 DNA ligase buffer; 

c. 24 uL H 2 0. 

Aliquot 14 [xL of the master mixture into each of four tubes. 

2. Add 5 uE of one of the dephosphorylated digestions (see Subheading 3.3.) to one of the 
above tubes and then add 1 uE of T4 DNA ligase (1 U/uE). Mix and incubate at room 
temperature for 1 h (see Note 3). One microliter of each ligation is then diluted to 50 fxL. 

3.5. PCR Amplification (BAC End Amplification) 

1. Prepare two reaction master mixtures. Add the following to a microcentrifuge tube on ice: 

a. 40 [xL 10X Taq Reaction buffer; 

b. 40 ixL 2 mM dNTP mix; 

c. 2 (xL primer Rsk; 

d. 3.2 (xL Taq polymerase; 

e. 312.8 uLH 2 0. 

Aliquot 199 [xL of the mixture into each of two microfuge tubes labeled F and R that will 
be called master mixture F and R, respectively. 

2. Add 1 fxL of primer Fbac or Rbac to master mixture F and R, respectively. Aliquot 50 (xL 
of the master mixture F into each of four empty tubes. 

3. Add 1 uE of one of the four diluted ligations (EcoRV, Hpal, Stul or Xmnl) to the tubes 
prepared earlier. The corresponding reactions are labeled EF, HF, SF, and XF. The PCR 
products from these tubes represent the left end of the BAC inserts. 

4. Correspondingly, aliquot 50 uE of the master mixture R into each of four empty tubes and 
add 1 fxL of one of the four diluted ligations. The PCR reactions are labeled ER, HR, SR, 
and XR. The PCR products from these tubes represent the right end of the BAC inserts. 

5. Overlay the reaction mixture with 50 (xL mineral oil. 

6. Using the following cycles, program the automated thermal cycler according to the 
manufacturers 's instructions (see Note 4): 

94°C (2 min), 1 cycle 

94°C (1 min), 62°C (1 min), 72°C (4 min) 30 cycles 
72°C (5 min) 1 cycle. 

7. Electrophorese 10 |xL from each reaction in an agarose gel in IX TAE buffer. An example 
of a typical result is shown in Fig. 2. 

8. Excise the amplification products from the gel and purify with the GenClean II kit. 



Isolation of BA C Ends 34 1 

M EF HF SF XF ER HR SR XR M 



Fig. 2. Agarose gel analysis of PCR products representing the terminal sequences of a B AC 
clone. EF, HF, SF, and XF represent PCR products obtained using ligations E, H, S, and X with 
primers Fbac and Rsk. ER, HR, SR, and XR represent PCR products obtained using ligations E, 
H, S, and X with primers Rbac and Rsk. For BAC clone A, a product representing the left end 
of the insert (T7 side) was obtained from PCR performed on ligations E and H, and a product 
representing the right end (SP6 side) was obtained from PCR performed on ligations E, H, and 
S. M, DNA size marker (k-Pst I (kb). 



4. Notes 

1. In this protocol, the four enzymes EcoRY, Hpal, Still, or Xmnl are chosen as the second 
enzyme of the double digestion, as these enzymes leave blunt termini after digestion. 
Actually, any enzyme that cuts frequently in the genomic DNA, but not in the left or right 
arm of the BAC vector can be used. However, the digested DNA should be ligated to the 
corresponding site of the anchor vector. Use of an enzyme that leaves a blunt terminus 
allows the user to prepare only one anchor vector that is compatible with all four enzymes. 
In very few cases, BAC ends cannot be obtained using the four enzymes recommended 
earlier. Other restriction enzymes that do not cut in the left arm or right arm, and fre- 
quently cut in the insert can be chosen as a second enzyme for the double digestion. The 
digested DNA should be ligated to the corresponding sites of the anchor vector. 

2. The plasmid pMSK that is prepared from pBluescript II SK(+) is used as an anchor vector 
to ligate with double digested BAC DNA. The majority of the LacL gene along with the 
T7 promoter sequence has been removed as these sequences will cause nonspecific PCR 
amplification. Actually, any plasmid with Noil and EcoRW (or any other enzymes leaving 
blunt termini) sites can be used as an anchor vector as long as the primer sequences in the 
anchor vector are not in common with the primers used to anneal to the BAC arms. 

3. In our lab, we usually prepare miniprep BAC DNA in the morning, and prepare PCR 
templates from the BAC DNA in the afternoon. The PCR can be run overnight, and BAC 
ends can be purified and labeled the second day. Usually, 1 \xL of miniprep BAC DNA is 



342 Yang and Mirkov 

sufficient as a source of template for the PCR amplification. If double-digested BAC 
DN A is ligated with the vector from 4 h to overnight, a higher quantity of the PCR prod- 
uct is obtained. As the DNA concentration from minipreps can vary, 2-5 \iL of the diluted 
ligation can be used as the PCR template. 
4. When using 1 u,L of the undiluted ligation as a PCR template, nonspecific amplification 
is frequently observed after 30 cycles of PCR, although a dominant amplification product 
can be observed that usually represents the BAC end. When ligations are diluted 1:100, 
and 1 |iL of the diluted ligation is used as the template for PCR, DNA fragments repre- 
senting the BAC end can also be obtained, except that the quantity of the amplification 
product is less abundant. In our protocol, we dilute the ligation 1:50. If DNA yield from 
the minipreps is low, ligations can be diluted 1:10 and 1 \iL of the diluted ligation can be 
used as the PCR template. Although this method was developed for BAC end isolation, it 
can also be used for YAC and PAC end isolation with some modifications. For PAC 
clones, the right arm (T7 side) has a. Notl site (4). Therefore, the right end can be isolated 
using the same protocol. The left arm of the PAC vector has a Sfil site which is an enzyme 
with 8-bp recognition sequence, and would be expected to cut infrequently in the genomic 
sequence. Thus, the left end can be isolated with double digestion of Sfi I and one of the 
EcoRW, Hpa\, Stul, and Xmnl digestions followed by PCR amplification. Primers should 
be designed accordingly. The primers specific for left ends or right ends of PAC clone 
should be designed based on the sequences of the left arm or right arm of the PAC vector. 
As for YAC clones, YAC2 has Notl and Sfil sites flanking the inserts (5). Thus, YAC 
ends can be isolated with this protocol except that Notl or Sfil is chosen based on the 
vector sequence. 

References 

1. Yang, Z. N. and Mirkov, T. E. (2000) Isolation of large terminal sequences of BAC inserts 
based on double-restriction-enzyme digestion followed by anchored PCR. Genome 43, 
412-415. 

2. Shizuya, H., Birren, B., Kim, U. J., Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M. 
(1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in 
Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89, 8794-8797. 

3. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory 
Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

4. Pierce J. C, Sauer, B., and Sternberg, N. (1992) A positive selection vector for cloning 
high molecular weight DNA by the bacteriophage PI system: improved cloning efficiency. 
Proc. Natl. Acad. Sci. USA 89, 2056-2060. 

5. Burke, D. T., Carle, G. F., and Olson, M. V. (1987) Cloning of large segments of exog- 
enous DNA into yeast by means of artificial chromosome vectors. Science 236, 806-81 1. 



36 



A "Step Down" PCR-Based Technique for Walking Into 
and the Subsequent Direct-Sequence Analysis 
of Flanking Genomic DNA 

Ziguo Zhang and Sarah Jane Gurr 
1. Introduction 

The hunt for missing sequence data, whether it be in pursuit of full-length clones or 
for promoter sequences, can be laborious and expensive. Indeed, protracted efforts to 
find missing sequences by library screening can be fraught with the frustration of for- 
aging through libraries that may lack the relevant clones. Such problems have led 
several researchers to describe alternative methods to clone and analyze DNA adja- 
cent to known sequences. 

Several polymerase chain reaction (PCR)-based methodologies are available for 
walking from a known region into cloned or uncloned genomic DNA, including inverse 
PCR (1,2), randomly primed PCR (3) and adaptor ligation (4-7). An improvement on 
the adaptor ligation method, combining "vectorette PCR" (6) with "suppression PCR" 
(9) was made (8) and, furthermore, an adaptor-ligation PCR protocol, using blocked 
adaptors and exonuclease digestion to remove unligated products, was described (10). 
More protocols followed, with a technique for the rapid acquisition of unknown DNA 
sequences by multiplex restriction site PCR (11), a method for the rapid amplification 
of genomic ends (12), and a technique to amplify and clone genomic DNA without 
restriction digestion (13). 

However, some of these PCR-based methods have proved to be complicated, ineffi- 
cient, or to demand genomic DNA of 50 kb in length as the starting material. In certain 
instances, not only can the collection of adequate amounts of biological tissues be 
troublesome, but so too can the preparation of high-molecular weight DNA. Indeed, this 
is our experience, as we work with a plant pathogenic fungus (barley powdery mildew) 
that is a true obligate parasite and that cannot, therefore, be grown axenically. 

We describe a novel and efficient PCR-based technique for walking into unknown 
flanking genomic DNA adjacent to a known sequence such as cDNA, without recourse 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

343 



344 Zhang and Gurr 

to protracted laborious library screening for overlapping sequences. This two compo- 
nent "hot start" and "step down" PCR method uses 6 X 1 jxg of genomic DNA (about 
20 kb in length) restricted with six different endonucleases and ligated to adaptors, 
with the inclusion of two further restriction enzymes to prevent self-ligation (14). It 
allows us to walk, in a single experiment, up to 6 kb into flanking DNA and gives us 
sufficient PCR products for up to 200 different walking experiments. The protocol is 
summarized diagrammatic ally in Fig. 1. 

2. Materials 

1. Genomic DNA: Many different methods describe the preparation of intact high-molecu- 
lar weight DNA. However, genomic DNA of quality adequate for Southern blot analysis 
is sufficient for this protocol. We routinely use DNA extracted from barley powdery mil- 
dew spores, prepared essentially as described for plant DNA extraction (15) using 
hexadecyltrimethylammonium bromide (CTAB). 

2. Restriction endonuclease enzymes: Spel, Avrll, Nhel, Xbal, Agel, Xmcil, NgoMlW, BspEl, 
and their respective buffers as supplied by New England BioLabs (NEB), Herts, UK. 
(see Notes 1 and 2). 

Group I restriction enzyme set the residual "ends": 

Spel: A/CTAGT — N 

*AvrII: C/CTAGG I I I 

Nhel: G/CTAGC — NGATC 

Xbal: T/CTAGA 
Group II 

*Agel: A/CCGGT — N 

Xmal: C/CCGGG I I I 

NgoMlV: G/CCGGC — NGGCC 

BspEl: T/CCGGA 

3. T4 DNA ligase by NEB. 

4. Primers and Adaptors, synthesized in MWG Biotech, AG Ebersberg, Germany. 

a. PP1: GTAATACGACTCACTATAGGGC 

b. PP2: ACTATAGGGCACGCGTGGT 

c. PPR1: P0 4 -CTAGGGCCACCACG-NH 2 

d. PPR2: P0 4 -CCGGTGCCACCACG-NH 2 (see Note 3) 

e. Padl: GTAATACGACTCACTATAGGGCACGCGTGG TGGCC 

f. Pad2: GTAATACGACTCACTATAGGGCACGCGTGGTGGCA 
The relationship between the synthesized primers and adaptors is: 
5'-GTAAT ACGAC TCACT ATAGG GCA CG CGTGG TGGCC 

*PP1 *PP2 I I I I I I I I I I 

H 2 N-GCACC ACCGGGATC-P0 4 -5' 
and named adaptor 1 (ADP1) 

5'-GTAAT ACGAC TCACT ATAGG GCACG CGTGG TGGCA 
PP1 PP2 I I I I I I I I I I 

H 2 N-GCACC ACCGTGGCC-P0 4 -5' 
and named adaptor 2 (ADP2) 

The nested primer sequences, PP1 and PP2, are underlined and italicized (see Note 4). 
The end of adaptor 1 (ADP1) shares sequence commonality with the group I restric- 
tion enzyme set. The end of adaptor 2 (ADP2) shares sequence commonality with the 
group II restriction enzyme set. End self-ligation is prevented by the addition of Avrll 
or Agel to the ligation buffer mix. 



"Step Down" PCR for Genomic Walking 

Genomic DNA 



345 



Restriction 

enzyme 

digestion 



V V V 

Spe I Nhe I Xba I 



JD, 



V V V 

Xmal NgoMIV BspE I 



Ligation to 
adaptors 
& further* 
restriction 

12°C-60min T. 
25°C-20min -* 
8°C-120 min 
70°C-6min 



Spe I 



V I 1 } 



Nhe I 



6 cycles 



V V V 

Xmal NgoMIV BspE] 
v .. ■> 



each ligation gives: 



Agel 



l-> -» I. 



=[ ^^ 



"Hot start" and "step down" 
PCR reaction 1 



PP2 

2T 



"Hot start" and "step down" 
PCR reaction 2 



"Step down" PCR: 

94°C-2sec V 
72°C-3min J 3 c y cles 

94°C-2sec "1 , 

7(TC-3min J 3 c y cles 

94°C-2sec V , 
68°C-3min | 3 C y cleS 

94°C-2sec "l 
66°C-20sec ["26 cycles 
68°C-3 min J 

68°C -8 min 



Direct sequence from both ends 

Fig. 1. A diagrammatic summary of the PCR strategy for walking into unknown flanking 
DNA. Genomic DNA was restricted with one of six different enzymes (Spel, Nhel, Xbal, Xmal, 
NgoMTV, BspEl), ligated to adaptors 1 and 2 and further restricted* by including in the reaction 
mix the initial enzyme (e.g., Spel) and also Avrll (set 1) or Agel (set 2). "Hot start" and "step 
down" PCR was undertaken in reaction 1 with primers PP1 and the gene-specific primers, PI, 
and again in reaction 2 with PP2, and the gene-specific primer, P2. 



346 Zhang and Gurr 

5. 10X PCR buffer: 400 mM Tricine-potassium hydroxide, 150 mM potassium acetate, 35 mM 
magnesium acetate, 37.5 jxg/mL bovine serum albumin, pH 9.2. 

6. 10X deoxynucleotide 5'-triphosphates (dNTPs): an aqueous solution of 2 mM of each 
dNTP deoxyadenosine 5'-triphosphate(dATP), deoxycytidine 5'-triphosphate (dCTP), 
deoxyguanosine 5'-triphosphate (dGTP), and deoxythymidine 5'-triphosphate (dTTP). 

7. Advantage cDNA polymerase mix, supplied by Clontech Laboratories, CA (see Note 4). 

8. Thermal cycler: Genius 96 wells cycler, supplied by Techne Ltd, Cambridge UK. 

9. PCR tubes: 0.2 mL thin wall 8 strip tubes, supplied by Anachem Ltd, Luton, UK. 
10. TE buffer: 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.6. 

3. Methods 

3. 1. Template Preparation 

3.1.1. Genomic DNA Restriction Enzyme Digestion 

1 . Digest six 1 u,g aliquots of barley powdery mildew DNA (about 20 kb in length) individu- 
ally with one of 6 restriction enzymes (Spel, Nhel, Xbal, Xmal, NgoMYV or BspEl, as 
described in Subheading 2.2., (see also Note 1) in a 50 |xL volume of their appropriate 
restriction enzyme buffer (Fig. 1). 

2. After 12 h digestion at 37°C extract the six separate DNA digestion samples twice in 
equal volumes of phenol/chloroform (v/v) and precipitate the DNA in sodium acetate/ethanol 
at-20°C (16). 

3.1.2. Adaptor Ligation: The Protocol is Summarized in Fig. 1 

1. Ligate the Spel, Nhel, and Xbal digested genomic DNA samples to adaptor 1 (PPR1 + 
Padl) and the Xmal, NgoMlY, and BspEl digested samples to adaptor 2 (PPR2 + Pad2). 
The six 20 uL volume ligation mixes are made up of ligation buffer (50 mM NaCl, either 
10 mM Tris-HCl (ligations 1-3) or 10 mM Bis. Tris-HCl (ligations 4-6), 10 mM MgCL, 
1 mM dithiothreitol (DTT), 1 mM adenosine triphosphate (ATP), pH 7.9 for ligations 
1-3; pH 7.0 for ligations 4-6), 1 [xg DNA, 100 pmole adaptor (ADP 1 or ADP 2), 6 U of 
T4 ligase, 6 U restriction enzyme Avrll (ligation set 1, 1-3), and 6 U initial restriction 
enzyme (e.g., Spel); or Agel (ligation set 2, 4-6) and 6 U initial restriction enzyme (e.g., 
Xmal) (see Notes 1 and 2). 

In summary, the 6 ligations set up are: 
Ligation 1: Spel digested genomic DNA, Avrll and Spel, adaptorl 
Ligation 2: A^el digested genomic DNA, Avrll and Nhel, adaptor 1 
Ligation 3, Xbal digested genomic DNA, Avrll and Xbal, adaptor 1 
Ligation 4, Xmal digested genomic DNA, Agel and Xmal, adaptor 2 
Ligation 5, NgoMYV digested genomic DNA, Agel and NgoMlV, adaptor 2 
Ligation 6, BspEl digested genomic DNA, Agel and B.?pEI, adaptor 2 

2. Place the ligation mixes in a Techne Genius PCR machine set to cycle between 12°C 
(60 min) and 25°C (20 min) for six cycles, followed by an 8°C for 120 min. 

3. Stop the ligation reaction by placing the tubes at 70°C for 6 min. 

4. Dilute the ligation mixes by the addition of 1 80 |xL TE buffer. 

5. Use 1 (xL of each diluted ligation mix to set the first round PCR amplification reaction. 

3.2. Primer Design 

Design and synthesize two nested primers (e.g., PI and P2) from the known 
sequence (see Note 3). 



"Step Down" PCR for Genomic Walking 347 

Any DNA sequence that is longer than 50 bp provides a suitable anchor from which 
to walk into adjacent unknown sequence. Wherever possible, each of the primers 
should be about 30 bases in length, with ideally, 10 to 15 GC residues. Unless unavoid- 
able, the primer pairs should not overlap with each other's sequence. 

3.3. PCR Amplification 

3.3. 1. First-Round PCR Amplification 

1 . Set up a 20 uL PCR mixture with 40 rail Tricine-potassium hydroxide, 1 5 mM potassium 
acetate, 3.5 rnM magnesium acetate, 3.75 ug/mL bovine serum albumin, pH 9.2, 200 \xM 
dNTP mix, IX Advantage cDNA polymerase mix, 10 pmole of each primer and 1 uL of 
the diluted ligation mix (5 ng DNA). 

2. Initiate the amplification reaction with a "hot start", which is automatically performed 
with the inclusion of the TaqStart antibody (Advantage cDNA polymerase mix,Clontech). 
Subsequently, utilize our "step down" PCR protocol with the primers PP1/P1 and PP2/P2. 

3. Set the PCR machine to cycle: 

94°C for 2 s and 72°C for 3 min, 3 cycles; 

94°C for 2 s and 70°C for 3 min, 3 cycles; 

94°C for 2 s and 68°C for 3 min, 3 cycles; 

94°C for 2 s, 66°C for 20 s, 68°C for 3 min, 26 cycles; 

68°C for 8 min. 

4. Dilute 5 u,L of the PCR products from first-round PCR amplification with 200 uL sterile 
distilled water. 

5. Use 1 uL of the diluted first-round PCR products in second-round of PCR amplification. 

3.3.2. Second-Round PCR Amplification 

Amplicons may or may not be visible following gel electrophoresis of the products 
of first-round PCR amplification. If no bands are present after round 1 do not discard 
the tubes, but proceed to second-round PCR amplification. If bands are present after 
round 1, do not be tempted to stop and sequence these products: it is essential to 
progress to second-round PCR amplification, which progresses essentially as the first 
PCR, but it exploits the primers PP2 (nested for PP1) and P2 (nested for PI). However, 
if bands are present after round 1, but absent after second-round PCR amplification, 
this may be because of an intron boundary / primer problem (see Note 7). 

1. Visualize the PCR products from first- and second-round reactions following electro- 
phoresis through a 1.0% agarose gel (see Note 5). 

2. Excise the dominant bands from the gel and sequence the products directly. 

Fig. 2 shows a typical picture of the gel electrophoresis profile of the amplicons 
after second-round PCR amplification. 

4. Notes 

1. The choice of the initial six restriction enzymes used to cut the powdery mildew DNA 
was determined by: 

a. the resulting average length of the restricted DNA (about 4 kb), which is a suitable 
length for PCR amplification; 

b. the commonalitity of sequence at the cuts ends, i.e., N/NGATC with Spel, Nhel, and 
Xbal and N/NGGCC with Xmal, NgoMIW, and BspEl. 



348 Zhang and Gurr 




5M hp 



Fig. 2. Gel electrophoresis profile of the amplicons from second-round PCR amplification 
(with primers PP2 and P2) of the six adaptor-ligated enzyme digests (Spel, Nhel, Xbal, Xmal, 
ATgoMIV, BspEl, loaded from left to right across the gel). 



2. The restriction endonucleases Avrll and Agel were reserved for the ligation step as both 
enzymes tolerate the conditions optimal for the ligation step. They were included to pre- 
vent self-ligation of the adaptors (Avrll for adaptor 1 and Agel for adaptor 2). The second 
series of restriction enzyme digestions with Spel, Nhel, Xbal, Xmal, NgoMlW, and BspEl 
is included to obviate problems with genomic DNA fragment self-ligation. The reactions 
were therefore performed at 12°C (to favor ligation) and 25 °C (to favor restriction diges- 
tion) and the resultant ligation products are / were restricted genomic DNA with adaptors 
1 or 2 at both ends. 

3. The -P04 and -NH 2 groups were added to the ends of PPR1 and PPR2 to prevent nonspe- 
cific 3' end elongation during the PCR. Without the -NH 2 modification, the 3' end of the 
PPR1 (or PPR2) would elongate using Padl (or Pad2) as template at the beginning of the 
cycling protocol. As a result, no target fragment would amplify — as Padl (or Pad2) car- 
ries PP1 and PP2 primer sequence, and as both ends of all the genomic fragments in the 
DNA "pool" from the template preparation carry the Padl (Pad2) sequence. Similarly, 
the -P0 4 group modification is essential to ensure that PPR1(2) ligates to the 3' end of the 
genomic DNA. Indeed, without the — 4 modification the PPR1(2) would be lost in the 
first denaturation step and the 3' ends of both strands of the genomic DNA fragments 
would elongate using Padl(2) as template. Both the -NH 2 and -P0 4 modifications are 
therefore pivotal to successful gene-specific amplification. Without them, there would be 
spurious and nonspecific amplification. 

4. Three additional steps in the protocol are designed to further restrict spurrious annealing 
and sequence amplification. They are as follows. 

a. The "hot start" technique that is automatically performed by the Advantage cDNA 
polymerase mix. 

b. A series of "step down" temperature cycles (72°C to 66°C, ramped by 2°C), which 
allows the preferential annealing of gene specific primer (PI or P2). Notably, in first- 
round PCR amplification, the PP1 primer anneal sequence remains unavailable until 
the gene specific primer (PI) anneals to the target sequence (i.e., the initial DNA 



"Step Down" PCR for Genomic Walking 349 

synthesis). The "step down" protocol ensures that this initial synthesis is as specific 
as is possible, 
c. The inclusion of the nested primer pair that is pivotal to the success of second-round 
PCR amplification. 

5. The visualization of the products of second-round PCR amplification, following gel 
electrophoresis, often reveals several amplicons to be present in different samples 
(Fig. 2). Select one amplicon of the approximate length of sequence you require; this 
will usually be adequate. However, if a sequence longer than 1 kb is needed, select two 
bands (about 500 to 1000 bp) to be sequenced by the gene-specific primer (e.g., P2) and 
select all bands of different length to be sequenced by PP2 primer. This may facilitate 
the rapid collection of all overlapping sequences and so avoid the necessity for further 
experimentation. 

6. The DNA isolated from each gel band is sufficient for up to eight sequencing reactions. 
Furthermore, each sequencing reaction can give clear readable sequence data up to 
about 800 bp. However, some of the longer amplicons can sometimes yield relatively 
poor data, with only 300-400 bp of readable sequence. Here, it is useful to design new 
primer pairs based on this preliminary sequence data and to use these either to 
resequence second-round PCR amplicon or to use them to reamplify the products of 
first-round PCR amplification. 

7. Amplification problems could be encountered using a gene-specific primer designed from 
cDNA sequence data, where the primer spans an intron-exon boundary. In this instance, 
we have successfully sequenced a DNA fragment, where the P2 primer sequence spans 
such a boundary, by sequencing an amplicon from first-round PCR amplification. Alter- 
natively, a further primer could be synthesized to avoid this problem area. 

8. Regions of genomic DNA that are rich in repetitive sequences may pose a problem for 
this protocol. We have had no experience of this problem. 

9. Theoretically, if a particular gene is distant from the restriction enzyme recognition sites, 
then amplification will not occur. How likely is this? Given that the average distance 
between recognition sites is 4096 bp and add this to our ability to walk some 6000 bp then 
the likelihood of one 6 bp recognition site being absent per 6000 bp is 23%. However, as 
six different restriction enzymes are used and six amplifications are performed per walk, 
then the likelihood of all restriction sites being absent is about 0.0 1 %. On a more practical 
level, in our lab we have performed 80 different walking procedures and have covered 
some 200 kb sequence (14,17) without a single failed attempt! 

10. Use a proofreading DNA polymerase to reduce errors in sequence amplication. Further- 
more, the direct-sequence analysis of the amplicons, rather than the adoption of cloning 
procedures, further limits sequence error. 

Acknowledgment 

This work was supported by BBSRC grant ASD 43/A06377 and BBSRC/ 
COGEME grant 43/IGF 12456. 

References 

1. Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse 
polymerase chain reaction. Genetics 120, 621-623. 

2. Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) A procedure for in vitro amplifica- 
tion of DNA segments that lie outside the boundaries of known sequences. Nucl. Acids 
Res. 16,8186. 



350 Zhang and Gurr 

3. Parker, J. D., Rabinovitch, P.S., and Burmer, G. C. (1991) Targeted gene walking poly- 
merase chain reaction. Nucl. Acids Res. 19, 3055-3060. 

4. Riley, J., Butler, R., Ogilvie, D., Finniear, R., Jenner, D., Powell, S., Anand, R., Smith, J. C, 
and Markham, A. F. (1990) A novel, rapid method for the isolation of terminal sequences 
from yeast artificial chromosome (YAC) clone. Nucl, Acids Res. 18, 2887-2890. 

5. Rosenthal, A. and Jones, D. S. (1990) Genomic walking and sequencing by oligo-cassette 
mediated polymerase chain reaction. Nucl. Acids Res. 18, 3095-3096. 

6. Lagerstrom, M., Parik, J., Malmgren, H., Stewart, J., Pettersson, U., and Landegren, U. 
(1991) Capture PCR: efficient amplification of DAN fragments adjacent to a known 
sequence in human and Yac DNA. PCR Meth. Appl. 1, 1 1 1-1 19. 

7. Jones, D. H.and Winistorfer, S. C. (1993) A method for the amplification of unknown 
flanking DNA: targeted inverted repeat amplification. Biotechniques 15, 894-904. 

8. Siebert, P. D., Chenchik, A., Kellogg, D. E., Lukyanov, K. A., and Lukyanov, S. A. (1995) 
An improved PCR method for walking in uncloned genomic DNA. Nucl. Acids Res. 23, 
1087-1088. 

9. Lukyanov, S. A., Gurskaya, N. G., Lukyanov, K. A., Tarabykin, V. S., and Sverdlov, E. D. 
(1994) Highly efficient subtractive hybridization of cDNA. Bioorgan. Chem. 20, 701-704. 

10. Padegimas, L. S. and Reichert, N. A. (1998) Adaptor ligation-based polymerase chain 
reaction-mediated walking. Analyt. Biochem. 260, 149-153. 

11. Weber, K. L., Bolander, M. E., and Sarkab, G. (1998) Rapid acquisition of unknown DNA 
sequence adjacent to a known segment by multiplex restriction site PCR. Biotechniques 
25,415-419. 

12. Cormack, R. S. and Somssich, I. E. (1997) Rapid amplification of genomic ends (RAGE) 
as a simple method to clone flanking genomic DNA. Gene 194, 273-276. 

13. Rudi, K.,Fossheim, T., and Jakobsen, K. S. (1999) Restriction cutting independent method 
for cloning genomic DNA segments outside the boundaries of known sequences. 
Biotechniques 27, 1170-1172, 1176-1177. 

14. Zhang, Z and Gurr, S. J. (2000) Walking into the unknown: a "step down" PCR-based 
technique leading to the direct sequence analysis of flanking genomic DNA. Gene 253, 
145-150. 

15. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Deidman, J. G., and 
Strahl, K. (Eds.), Current Protocols on Molecular Biology, 1987. Wiley, NY, p. 233. 

16. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory 
Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY. 

17. Zhang, Z. and Gurr, S. J. (2001) Expression and sequence analysis of the Blumeria 
graminis mitogen-activated protein kinase genes, mpk\ and mpkl. Gene 266, 57-65. 



Library Construction and Screening 



37 

Use of PCR in Library Screening 

An Overview 
Jinbao Zhu 

1. Introduction 

Traditionally, libraries are screened with different probes to isolate target genes or 
sequences. These probes can be a particular sequence such as a cDNA, a polymerase 
chain reaction (PCR) product, or a genomic fragment (1). An oligonucleotide can be a 
probe if no closely related cDNAs or gene clones are available to use, or if only the 
amino acid sequence of a protein from a gene is available and the gene sequences can 
be derived by reverse-translating (2,3). Sequences derived from mRNA can also be 
used as a probe to identify gene sequences according to the differences in their con- 
centrations from different biological sources (1). In addition, antibody probes can also 
be used to isolate cDNA clones for target proteins (4). However, using cDNA, PCR 
product and gene probes is limited in library screening depending on the availability 
of a sequence of sufficient similarity to cross-hybridize with a clone containing the 
target gene (7). In screening a library with a nucleic acid probe, plates are prepared 
with either bacterial colonies or with plagues from a phage library (7). The general 
screening procedure, for example, in screening cDNA in phage, includes titering the 
library to determine the phage concentration; plating and lifting the phage particles; 
hybridizing the immobilized cDNA with appropriate radiolabeled DNA probes; and 
selecting positive plaques by autoradiography. The procedure is usually cycled in order 
to isolate a plaque with a single cDNA. This procedure, however, can be time-con- 
suming, labor-intensive, and difficult with requirements such as radiolabeling. Today, 
the PCR has become a significant technique in identifying and isolating positive clones 
from libraries. With the sensitivity and specificity of PCR technique, the advantages 
of PCR-based library screening can be recognized: 1. False positive clones can be 
avoided since positives are identified by DNA bands of appropriate size; 2. Overall 
screening time can be shortened; 3. Screening of multiple genes with appropriate prim- 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

353 



354 Zhu 

ers can be performed in the same PCR (5). As a result, PCR has been extensively used in 
the screening of cDNA or genomic libraries (2,6-10) or large-insert DNA libraries of 
bacterial artificial chromosome (BAC) (11-13) and yeast artificial chromosome (YAC) 
(14-16). 

There are many applications of using PCR techniques in library screening and some 
applications are reviewed as follows. 

2. cDNA and Genomic Library Screening 

Narayana R. Isola et al. (8) described a rapid and less expensive method to screen 
cDNA libraries utilizing PCR to amplify target sequences from the libraries. In study- 
ing the expression of H-CAM gene family, they designed oligonucleotide primers to 
amplify a region of the apparently conserved, putative extracellular domain to simplify 
and more efficiently identify the presence of CD44 transcripts (17) within cDNA 
libraries from a variety of tissues and cells. With the screening technique, a novel 
CD44 transcript was isolated from a reticulocyte cDNA library. And the total proce- 
dure could be accomplished in three days (8), This method also can be used to isolate 
multiple members of a protein family as well as homologous genes in different species 
by designing appropriate primers to amplify the most conserved regions (8). 

Bloem and Yu (6) introduced a time-saving method to screen cDNA or genomic 
libraries using PCR, which eliminates the time consuming steps of filter hybridization. 
Briefly, plating was made at the density of 50,000 pfu/150 mm plate. Then 20 u,L filter 
rinse was used as the template for PCR and followed by agarose gel electrophoresis to 
determine if the plate had phage plaques with the target sequence by comparing to the 
DNA band of appropriate size in the gel. Filtering was then made on the target plate 
and the filter along with the agar was cut into different sections. PCR was then per- 
formed on each section and the PCR products were checked by agarose gel electro- 
phoresis. The target phage could be isolated further by more plating and PCR or plating 
and filter hybridization. The method was simplified with only one round of plating 
and two orders of PCR before any filter hybridization. With this procedure, multiple 
pairs of primers can be used in the same PCR reaction to isolate several genes/frag- 
ments at the same time. 

Similar to filter hybridization, plague hybridization is labor-intensive, and often 
creates false positive recombinant clones in screening genomic or cDNA fragments 
wrapped within phage libraries (9). As an alternative, David I. Israel (9) described a 
PCR-based screening method that improved the specificity of the screening by requir- 
ing that three oligonucleotides (two PCR primers and one hybridization probe) cor- 
rectly annealed to the desired gene fragment and decreased the odds of acquiring false 
positive clones. In this protocol, the initial genomic library in A, phage was subdivided 
into 64 wells. PCR was then performed on the pooled amplified phage using specific 
oligonucleotide primers. A single well with the target gene sequence was identified 
through a PCR product of the right size that hybridized with an internal oligonucle- 
otide probe. The positive phage was then used to infect bacteria at a lower number of 
phage particles per well for a secondary screening, and followed by a subsequent ter- 
tiary screening using the same protocol. This protocol can be applied to screen phage 



PCR in Library Screening 355 

and plasmid libraries of all types and for all genes as long as sequence information is 
available for designing the appropriate primers and an internal hybridization probe. 

According to James W. Larrick (10), using quantitative reverse transcription 
(RT)-PCR to screen cDNA libraries is an extremely sensitive technique, which will 
find wide application in biomedical research and diagnostics for cancer, metabolic 
diseases, and autoimmune diseases. In neurobiology, the technique can be used to 
dissect complex immunological processes and it may also be used to examine func- 
tional aspects of the nervous system; in oncology, it can be used to detect the expres- 
sion of abnormal genes using very small quantities of tumor tissue and studying biopsy 
material of limited availability; in virology and infectious diseases, the technology 
provides a useful tool in the study of the pathogenesis of viral diseases, particularly to 
study the expression and kinetics of viral proteins as well as the host cell's response to 
proteins such as cytokines, interferons, and stress proteins. RT-PCR also can be used 
to create a cDNA probe that can be used for library screening. For example, Caruso et al. 
(18) obtained a specific cDNA probe by RT-PCR and screened a cDNA library. 

Chiang et al. (7) recently studied transcription patterns of sequences on human chro- 
mosome 21 using PCR to screen embryonic, fetal, and adult human cDNA libraries. 
Seventy-three primer pairs were used to screen 41 different cDNA libraries using prim- 
ers previously used to screen a fetal brain cDNA library. They concluded that their 
data indicated significant overlap between the genes expressed in different tissues, 
which is consistent with the supposition that there are as few as 60,000-70,000 human 
genes. They indicated that PCR-based cDNA library screening technique was easier to 
perform than Northern blots or RNase protection assays although large numbers of 
primers might be required to be designed and synthesized. 

3. YAC Library Screening 

In screening of yeast artificial-chromosome (YAC) clones, colony hybridization 
was commonly used in identifying target sequences of genes (19). However, screen- 
ing of YAC libraries by colony hybridization causes certain problems according to 
Heard et al. (15), and becomes cumbersome with a large scale of screening (14). The 
problems are: 1. The requirement of primary yeast transformants must be implanted in 
agar; 2. The number of YACs per cell and the number of cells per yeast colony are 
much lower than in cloning in bacteria; 3. The spheroplasting of the colonies on the 
filters prior to lysis can be inefficient leading to under-representation of some colonies 
(14). However, YAC library screening utilizing PCR has been proved to be rapid, 
sensitive and efficient. For example, Heard et al. (14) described a method in which 
YAC clones were screened using PCR techniques. In brief, 96 colonies were obtained 
from primary yeast transformants by growing on 96 well microtitre dishes and then 
followed by several steps to obtain the DNA for screening. For screening, 1 u,L of 
DNA from each of the pools was used in a single PCR using a pair of oligonucleotide 
primers derived from a known target sequence. A positive colony could be identified 
with a band of appropriate DNA size. 

Green and Olson (14) developed an approach for screening large, ordered libraries 
of YAC clones based on PCR techniques with sequence tag site (STS) content map- 



356 Zhu 

ping. The method starts with individual clones growing in arrays of 384 colonies per 
nylon filter. The general strategy for screening human YAC libraries includes two 
consecutive PCR after pooling single-filter pools containing 384 human- YAC clones. 
The first PCR is performed on multifilter pools to identify the positive target pools. 
The second PCR is made on each of the constituent single-filter pools from the posi- 
tive multifilter pools. If a single-filter pool is detected with the appropriate PCR prod- 
uct as separated in polyacrylamide gels and detected by ethidium bromide staining, 
the positive clone in the 384-clone array can be located by colony hybridization using 
the probe of the radiolabeled PCR product. With this strategy, a 23,000-clone YAC 
library for several single-copy human genes has been successfully screened. The PCR- 
based approach offers several advantages over colony hybridization methods. For 
instance, the high sensitivity and specificity of this technique can allow a single posi- 
tive clone to be screened using only 100 ng of DNA from a pool representing thou- 
sands of YAC clones. With this protocol, it is possible to determine if a particular 
DNA sequence is present in a YAC library in less than a day. However, the limitation 
of this method might be the requirement of DNA sequence and extensive specific 
primer synthesis. As suggested by the authors, this protocol could be further improved 
by eliminating the colony hybridization in the second stage for better overall effi- 
ciency. For example, Kwiatkowski et al. (16) described a PCR protocol for screening 
single filters of matrix pools eliminating the step of colony hybridization, and intro- 
duced a method for PCR of crude yeast ly sates. 

4. BAC Library Screening 

Asakawa et al. (72) reported a protocol to screen a human genomic BAC with a 
two-step PCR. The first PCR screening was applied against ten superpools of 9600 
BAC clones. The second screening was employed to DNA samples achieved by four- 
dimensional PCR (4D-PCR) for an identified superpool. The two-step PCR screening 
could allow isolating a desired BAC clone(s) within a day or so. However, according 
to the authors, only one positive clone should be present in a given superpool on the 
4D or multidimensional two-step PCR screening procedure. If more than one positive 
clone (n a 1) were detected in a single superpool, third screening step with at most n 4 
PCR assays would be required. In comparison to a 3D-PCR screening procedure (13), 
the 4D-system might be simpler with less PCR reactions. However, the 3D-screening 
procedure (13) had the advantage of avoiding a third screening (11). Two-dimensional 
PCR was also used to screen BAC library. For example, Crooijmans et al. (12) 
illustrated a protocol using two-dimensional PCR with 125 microsatellite markers and 
successfully screened a chicken BAC library. 

Xu et al. (20) described a protocol using arbitrary primers (AP-PCR) to screen 
overlapping BAC libraries. They used a rice BAC library to prepare the pools of BAC 
DNA and chose 22 arbitrary primers to examine the pooled BAC DNAs and indi- 
vidual BAC DNAs. Each primer identified 1-10 loci with the average of 4.4 loci. A 
total number of 245 overlapping BAC clones was identified and confirmed by DNA- 
DNA hybridization. This method takes the advantages of Green and Olson's method 
(14) but eliminates the need of synthesizing specific primers. 



PCR in Library Screening 357 

The PCR technique has dramatically improved library screening compared to filter 
or plague hybridization. In addition to simplifying the procedure, PCR has increased 
the specificity and efficiency of library screening. The disadvantages for screening 
using PCR technique might be the availability of sequence information and prepara- 
tion of oligonucleotide primers. This drawback, however, has been diminished with 
the development of DNA sequencing techniques and availability of DNA sequences. 
The relevant detailed PCR-based screening protocols will be presented in the follow- 
ing chapters of this section. 

References 

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tion: Principles and Practice, Foster, G. D. and Twell, D. Eds., Wiley, New York, pp. 
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7. Chiang, P. W., Kurnit, D. N., Osemlak-Hanzlik, M., and Trbusek, M. (1995) Rapid PCR- 
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8. Isola, N. R., Harn, H. J., and Cooper, D. L. (1991) Screening recombinant DNA libraries: 
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9. Israel, D. I. (1993) A PCR-based method for high stringency screening of DNA libraries. 
Nucl. Acids Res. 21, 2627-2631. 

10. Larrick, I. W. (1992) Message amplification phenotyping (MAPPing)-principles, practice 
and potential. Trends Biotechnol. 10, 146-152. 

11. Asakawa, S., Abe, I., Kudoh, Y., Kishi, N., Wang, Y., Ryo, K., Kudoh, I., Kawasaki, K., 
Minoshima, S., and Shimizu, N. (1997) Human BAC library: construction and rapid 
screening. Gene 191, 69-79. 

12. Crooijmans, R. P., Vrebalov, I., Dijkhof, R. I., van der Poel, I. I., and Groenen, M. A. 
(2000) Two-dimensional screening of the Wageningen chicken BAC library. Mamm. 
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13. Kim, U. I., Birren, B. W., Slepak, T, Mancino, V., Boysen, C, Kang, H. L., Simon, M. I., 
and Shizuya, H. (1996) Construction and characterization of a human bacterial chromo- 
some library. Genomics 34, 213-218. 

14. Green, E. D. and Olson M. V. (1990) Systematic screening of yeast artificial-chromosome 
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358 Zhu 

15. Heard, E., Davis, B., Feo, S., and Fried, M. (1989) An improved method for the screening 
of YAC libraries. Nucl. Acids Res. 17, 5861. 

16. Kwiatkowski, T. J., Zoghbi, H. Y., Ledbetter, S. A., Ellison, K. A., and Chinault, A. C. 
(1990) Rapid identification of yeast artificial chromosome clones by matrix pooling and 
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17. Harn, H. J., Isola, N. R., and Cooper, D. L. (1991) The multispecific cell adhesion mol- 
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18. Caruso, C, Bertini, L., Tucci, M., Caporale, C, Leonardi, L., Saccardo, F., Bressan, R. A., 
Veronese, P., and Buonocore, V. V. (2000) Isolation and characterization of wheat cDNA 
clones encoding PR4 proteins. DNA Sec/. 10, 301-307. 

19. Brownstein, B. H., Silverman, G. A., Little, R. D., Burke, D. T., Korsmeyer, S. J., 
Schlessinger, D., and Olson, M. V. (1989) Isolation of single-copy human genes from a 
library of yeast artificial chromosome clones. Science 244, 1348-1351. 

20. Xu, J., Yang, D., Domingo, J., Ni, J., and Huang, N. (1998) Screening for overlapping 
bacterial artificial chromosome clones by PCR analysis with an arbitrary primer. Proc. 
Natl. Acad. Sci. USA 95, 5661-5666. 



38 

Cloning of Homologous Genes by Gene-Capture PCR 

Renato Mastrangeli and Silvia Donini 

1. Introduction 

Conventional procedures to isolate a gene belonging to an ortholog family usually 
imply the use or the construction of double-stranded cDNA libraries derived from a 
specific mRNA source of interest (cells or tissues) (1). The double-stranded DNA 
library plated on various membranes is screened by filter hybridization with a radiola- 
beled probe (1) derived from the known homologous gene. Clones or plaques hybrid- 
izing with the probe are then isolated and sequenced to find the gene of interest. This 
approach is time-consuming and a large number of false positive clones might be 
obtained, given that no homology is a priori available, especially when the homolo- 
gous probe contains a short and stable stretch of a sequence sharing clustered homol- 
ogy with both strands of the cDNA library. Alternatively, homologous genes may be 
isolated by PCR, but only when specific primers are available. 

Genomic sequence data are now available from a limited number of eukaryote 
model organisms (2-6). However, this number will increase in the near future (7,8) 
and bioinformatics will provide the easiest and fast way to search homologous genes. 
Meanwhile, data derived from the sequenced genomes and from differential gene 
expression analysis are making available a huge number of genes that have to be func- 
tionally characterized. To assess the gene function and to study phylogenetic relation- 
ships in various model organisms, screening procedures to search for gene families 
(orthologs and paralogs) are still necessary when the sequencing data are not available 
for the organism of interest. 

Gene-Capture polymerase chain reaction (PCR) or (GC-PCR) is a cloning strategy 
successfully used to isolate the full-length coding cDNA sequence of the mouse 
ortholog of human lymphocyte activation gene 3 (LAG-3) from total RNA of mouse 
activated thymocytes (9). The method is based on PCR amplification of single-stranded 
cDNA molecules selectively isolated from a starting cDNA mixture by multiple and 
sequential capture steps driven by magnetic beads bound homologous probes (capture 
probes). Two capture steps with biotinylated human homologous probes and 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

359 



360 Mastrangeli and Donini 

streptavidin-coupled magnetic beads were sufficient to isolate the mouse LAG-3 
cDNA, which was found to be 79% homologous to the human cDNA (9). 

The strategy of GC-PCR is shown in panels A-D of Fig. 1. The starting single- 
stranded cDNA library must contain suitable sequences at the 5' and 3' ends, to fur- 
ther allow PCR amplification (see panel A and D). This mixture contains all the 
reverse cDNA molecules of the library with 3' modified by homopolymeric tailing 
(see panel A). 

The capture probe is a biotinylated forward single-stranded DNA molecule (a syn- 
thetic oligonucleotide or a cDNA fragment prepared by PCR) linked to streptavidin- 
coupled magnetic beads. Sequences of the various homologous capture probes are 
derived from nonoverlapping encoding regions of the gene of interest (see panel B). 

A capture step consists of a fast liquid-phase hybridization reaction (first-order 
kinetic) between a single-stranded cDNA mixture and a high concentration of single- 
stranded homologous capture probe followed by magnetic separation of hybridized 
(captured) single-stranded cDNA molecules. All cDNA molecules forming stable 
complexes with the capture probe will be retrieved from the solution containing the 
high abundance of nonhybridizing cDNA molecules. The selection of full-length 
encoding cDNA molecules is performed by repeated capture steps with different 
homologous capture probes on the single stranded cDNA molecules released in the 
previous capture step. Only long cDNA molecules forming stable complexes with 
the new capture probe will be retrieved from the solution containing the nonspecific 
single-stranded cDNA molecules that hybridized in the previous capture step. 
Shorter cDNA molecules of the target gene derived from incomplete retrotran- 
scription and/or partially degraded RNA, and lacking the hybridizing region will be 
left in solution (see panel C). 

The capture steps allow the easy and fast magnetic separation of DNA complexes 
bound to magnetic beads from a complex cDNA mixture of the target source, when a 
magnet is applied (see panel C). The high sensitivity of PCR allows then the amplifi- 
cation of the minute amounts of captured and released cDNA molecules. The use of 
one base anchored oligo dT mix in both RT reaction and in the first PCR cycle of 
captured molecules (see panel A and D) allows the synthesis of specific cDNA 
fragments with homogeneous terminal ends (10). Southern blot analysis of the 
amplified cDNA molecules will help to identify and isolate the fragments correspond- 
ing to the full-length target gene. Sequencing of the isolated cDNA will confirm the 
success of the applied procedure. Two independent PCR amplifications followed by 
DNA sequencing must be performed to unequivocally determine the DNA sequence 
of interest (see panel D). 

Compared to conventional library screening GC-PCR represents a sensitive, fast, 
simple, specific, and safe method to easily isolate full-length ortholog genes. The start- 
ing material is a small amount of total RNA extracted from the cell or tissue of inter- 
est. This turns out to be useful when a small RNA source is available. The starting 
amplifiable single-stranded cDNA library has less complexity when compared to 
double-stranded cDNA libraries. The sequential hybridization with different probes 
performed on single-stranded cDNA molecule allows a further high-complexity 



Homologous Gene Isolation by GC-PCR 



361 



-► AAAAAAAAAAAA 



I 



illinium ] 



RT 



4 




fc. AAAAAAAAAAAAA 

mini i 


^ 


I 


RNase H 


^ — 


■■■■■■■■■ill I 



| 1) Microcon 100 
I 2)TdT+dATP 



mRNA (in total RNA) 

Oligo dT mix 
(AT17A+AT17C+AT17G) 



mRNA 
cDNA 



CDNA 



AAAAAAAAAAAAA -^" 



lllllllllll 



poly(A)-tailed 

single stranded cDNA 



B 



BFP3 



jB _FP2 



BFP1 



, m m m M m^^ 




RP3 RP2 

Known homologous gene as DNA template 



RP1 



PCR with primer pairs 

BFP1/RP1, BFP2/RP2 and BFP3/RP3 




BFP3/RP3 BFP2/RP2 BFP1/RP1 

Biotinylated double stranded homologous probes 




SV/B . 



1 



'SV (SV-magnetic beads) 

•SV/B . 



BFP3/RP3 BFP2/RP2 BFP1/RP1 

Immobilized double stranded homologous probes 




'sv/i 



I 




Alkali denaturation 

•SV/B 

CP3 " CP2 ""' CP1 

Immobilized single stranded homologous probes 

Fig. 1. (A) Synthesis of amplifiable single-stranded cDNA. (B) Synthesis and immobiliza- 
tion of single-stranded homologous probes CP1, CP2, and CP3 on magnetic beads. 



362 



Mastrangeli and Donini 



Poly(A)-tailed single stranded cDNA+ CP1 
Hybridization reaction 



First capture step 





CP1 




nnn 



Full-length target homologous cDNA 
CP1 



— /^/^ 



nrm 



AAAAAAA 



Incomplete target homologous cDNA 
CP1 



y^ 



Non specific cDNA molecules 



nnn 

flecules 



AAs 



AAAAAA-O 

(JVnnn 



Non-hybridizing 
cDNA molecules 



CP1 captured and released Poly(A)-tailed single stranded cDNA+ CP2 
Hybridization reaction 




Second capture step 



AAAAAAA- 




AAAAAAA 



nnn 

ncomplete target homologous cDNA 

nnn 



Full-length target homologous cDNA 

CP2 AAAAAAA ^ 



AAAAAAA 1SSS> 

INI 



j^ nnn 



^ IHI I | Non specific cDNA molecules 



Incomplete target homologous cDNA 



Non-hybridizing 
cDNA molecules 



CP1+CP2 captured and released Poly(A)-tailed single stranded CDNA+CP3 
Hybridization reaction 



I 



Third capture step 



0) 

c 

CO 

2 



CP3 

AAAAAAA-\/<JjJilj"~\_ 




AAAAAAA 



;ornple 




Full-length target homologous cDNA 



Incomplete target homologous cDNA 



Non-hybridizing 
cDNA molecules 



Fig. 1. (C) Selection by capture steps of full-length target homologous cDNA. 



Homologous Gene Isolation by GC-PCR 363 

Q CP1+CP2 captured and released poly(A)-tailed single stranded cDNA 



I 



Second strand cDNA synthesis 
primed with oligo dT mix 



Oligo dT mix 




Full-length target homologous cDNA 

Oligo dT mix 

imn 



AAAAAAAA- 

Incomplete target homologous cDNA 

PCR with AP primer 




AP 



I 



iiiiii i 





1 1 


Full-length target homologous cDNA 


AP 


AP 
i i 


► 




< a 

Incomplete target homologous cDNA AP 



EtBr Stained gel Southern blot with CP3 probe 




Full-length target homologous cDNA 



Incomplete target homologous cDNA 
and non specific cDNA 



Sequence confirmation 



Isolation and cloning 



Sequencing 



Fig. 1. (D) PCR, Southern analysis, and sequencing of selected single-stranded cDNA. 



364 Mastrangeli and Donini 

reduction in each capture step, resulting in a strong reduction of the hybridization 
background. In addition, the hybridization reaction is performed in a microcentrifuge 
tube, thus allowing the simultaneous processing of many samples. cDNAs from dif- 
ferent libraries (source and/or species) may be therefore easily analyzed. 

The method has the potential to isolate genes sharing conserved regions of suitable 
length, gene variants, and gene-encoding proteins with only limited knowledge of their 
amino acid sequence. Finally, GC-PCR may also work on DNA libraries (11). 

2. Materials 

Use molecular biology grade reagents. All described oligonucleotides may by pur- 
chased from any DNA Synthesis vendors or synthesized in house as described (11). 

2.1. Extraction of Total RNA and Synthesis of Amplifiable cDNA 

1. TriZol reagent (Life Technologies, Gaithersburg, MD). 

2. RNase-free water. 

3. Agarose gel electrophoresis reagents and equipment for RNA analysis. 

4. 30 u.M Oligonucleotide Anchored T 17 G (AT17G) in RNase-free water: 5'GGCCCTG- 
G ATCCGG ACCT AATTTTTTTTTTTTTTTTTG3 ' . 

5. 30 u.M Oligonucleotide Anchored T 17 A (AT17A) in RNase-free water: 5'GGCCCTG- 
G ATCCGG ACCT AATTTTTTTTTTTTTTTTT A3 ' . 

6. 30 \iM Oligonucleotide Anchored T 17 C (AT17C) in RNase-free water: 5'GGCCCTG- 
GATCCGGACCTAATTTTTTTTTTTTTTTTTC3'. 

7. 10 uM Oligo dT mix : 30 uM AT17G + 30 uM AT17A + 30 uM AT17C (1:1:1). 

8. RNase H (Roche Diagnostics). 

9. RNase inhibitor (Roche Diagnostics). 

10. Superscript™ II RNase H" Reverse Transcriptase (Life Technologies). 

11. 0.1 M Dithiothreitol (DTT). 

12. 10 mM Deoxynucleotide 5'-triphoshate (dNTP). 

13. 0.2X TE buffer: 2 mM Tris-HCl, 0.2 mM ethylenediaminetetraacetic acid (EDTA), pH 8 .0. 

14. Microcon 100 (Millipore, Beverly, MA). 

15. TdT buffer: 200 mM Tris-HCl pH 8.4, 500 mM KC1, 25 mM MgCl 2 , 1 mg/mL BSA. 

16. Terminal deoxynucleotide transferase (TdT) (Life Technologies). 

17. 2.5 mM Deoxyadenosine 5'-triphosphate (dATP) (see Note 1). 

2.2. Synthesis and Immobilization of Biotinylated Capture Probes 
to Magnetic Beads 

1. DNA source containing the known cDNA of interest (user). 

2. Oligonucleotides designed by the user: 

a. 5'-Biotinylated forward primer 1 (BFP1). 

b. 5'-Biotinylated forward primer 2 (BFP2). 

c. 5'-Biotinylated forward primer 3 (BFP3). 

d. Reverse primer 1 (RP1). 

e. Reverse primer 2 (RP2). 

f. Reverse primer 3 (RP3). 

3. Pfu DNA polymerase (Stratagene, La lolla, CA). 

4. 10X Pfu buffer provided with the enzyme. 

5. Glycerol. 

6. 2.5 mM dNTP. 



Homologous Gene Isolation by GC-PCR 365 

1 . Agarose gel electrophoresis reagents and equipment. 

8. TEN buffer: 10 mM Tris-HCl, 0.1 mM EDTA, \M NaCl, pH 8.0. 

9. Dynabeads M-280 Streptavidin 10 mg/mL (Dynal, Oslo, Norway). 

10. Magnetic particle concentrator for Eppendorf microtubes (Dynal MPC-E)(Dynal, Oslo, 
Norway). 

11. 0.15MNaOH. 

12. OLIGO Primer Analysis Software (National Bioscience, Inc., Plymouth, MN). 

2.3. Capture Step 

1. 20X SSC: 3 M NaCl, 0.3 M sodium citrate. 

2. 10% (w/v) sodium dodecyl sulfate (SDS). 

3. Hybridization and washing buffer: 6X SSC, 0.1% (w/v) SDS. 

4. Washing buffer: IX SSC, 0.1% (w/v) SDS. 

5. IX TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. 

6. DNase-free BSA 20 mg/mL (Roche Diagnostics). 

7. Hybridization oven and water bath. 

2.4. DNA Amplification 

1 . Reagents and equipment for PCR. 

2. Thermostable DNA polymerases: 

a. Taq DNA polymerase (Advanced Biotechnologies Ltd., Leatherhead, Surrey, UK). 

b. Pfu DNA polymerase (Stratagene, La Jolla, CA). 

3. 10X Taq buffer (without MgCL) (buffer IV, vial 1 provided with Taq enzyme). 

4. Dimethyl sulfoxide (DMSO). 

5. 25 mM MgCl 2 (buffer IV, vial 2 provided with Taq enzyme). 

6. 6.5mMdNTP. 

7. Anchor primer (AP): 5' GGCCCTGGATCCGGACCTAA 3'. 

2.5. Reagents for Southern Blot Analysis 

1. PCR DIG Labeling Mix (Roche Diagnostics). 

2. Blocking Reagent (Roche Diagnostics). 

3. Positively charged nylon membrane (Roche Diagnostics). 

4. DNA Molecular Weight Markers and digoxigenin-labeled DNA Molecular Weight Mark- 
ers (Roche Diagnostics). 

5. Hybridization buffer: 5X SSC, 0.1% (w/v) SDS, 1% N-lauryl sarcosine, and 0.5% (w/v) 
Blocking reagent. 

6. 5X SSC; 2X SSC; and IX SSC,0.1%SDS. 

7. Buffer A: 100 mM maleic acid, 150 mM NaCl, pH 7.5. 

8. Buffer B: 1% Blocking reagent in Buffer A. 

9. Anti-Digoxigenin-POD, Fab fragments (Roche Diagnostics). 

10. 100 mMTris-Cl, 150 mM NaCl, pH 7.5. 

1 1. Washing buffer: 0.3% Tween 20 in buffer A. 

12. ECL reagents (Amersham Pharmacia Biotech). 

13. Photogene development folder (Life Technologies) or SaranWrap. 

14. Hyperfilm ECL (Amersham Pharmacia Biotech). 

2.6. DNA Cloning and Sequencing 

1. Conventional cloning reagents and equipment. 

2. Reagents and equipment for DNA sequencing. 



366 Mastrangeli and Donini 

3. Method 

3.1. Synthesis of Amplifiable Single-Stranded cDNA 
Starting from Total RNA 

3.1.1. Total RNA Extraction 

1. Extraction of total RNA from cells or tissues is performed with TriZol reagent, according 
to the manufacturer's instructions; alternatively, other conventional procedures might be 
used. The RNA preparation should be of good quality. 

2. At the end of the procedure, dissolve total RNA in RNase-free water and store at -20°C. 

3.1.2. Reverse Transcriptase (RT) Reaction 

Total RNA (5 fxg) is reverse transcripted in 0.5-mL Eppendorf tube (see Note 2). 

1. Add a volume corresponding to 5 u.g total RNA, then 2 u,L oligo dT mix (10 u,M each 
primer), and RNase-free water up to 10 u.L total volume. 

2. Incubate at 65°C for 10 min, then chill on ice. 

3. Spin down the liquid and add the following components: 10 uL of 5X Superscript II 
buffer, 5 uL 0.1 M DTT, 5 uL 10 mM dNTP, 1 uL RNase inhibitor (10-50 U), 2 u,L 
Superscript II (400 U), 17 |xL RNase-free water. Total volume is 50 uL. 

4. Incubate samples at 37 C C for 90-120 min. 

5. Stop the reaction by incubating at 90°C for 5 min, then chill on ice for 10 min. 

6. Add 1 uL RNase H (1 U) and incubate samples for 30 min at 37 C C to degrade RNA. 

7. Add 0.2 mL of 0.2X TE buffer and transfer the resulting solution to the sample reservoir 
of a Microcon 100. Insert into a microcentrifuge, ultrafiltrate at 500 x g for 5 min follow- 
ing Microcon instructions. Four more ultrafiltration steps are then performed with 0.5 mL 
of 0.2X TE buffer at 500g for 12 min. Final volume is adjusted to 50 uL with 0.2X TE 
buffer (see Note 3). 

8. Store single-stranded cDNA at -20°C. 

3.1.3. Synthesis of Amplifiable Single-Stranded cDNA 
by Poly (A) Tailing with TdT 

In this step the synthesized reverse single-stranded cDNA is modified to allow PCR 
amplification. Many procedures may be used, each procedure has its own advantages 
in terms of simplicity, cost, rapidity (see Note 4). Here, the simple TdT homopoly- 
meric tailing with dATP is used. A poly (A) tail is added to the 3' end of the reverse 
strand of single-stranded cDNA. This allows the same primer mixture (oligo dT mix) 
to be used in the RT reaction and for the second-strand cDNA synthesis (see Note 5). 

1. Transfer 16 uL of single-stranded cDNA mixture (corresponding to 1.6 u.g total RNA) in 
a 0.5-mL Eppendorf tube. Incubate at 70°C for 5 min, then chill on ice. 

2. Spin down the liquid and add the following components: 1 uL TdT buffer, 2 u,L dATP 
(2.5 mM), 1 uL TdT (10 U). Total volume is 20 uL. Incubate at 37°C for 10 min (see Note 1). 

3. Inactivate TdT by incubating at 70°C for 10 min. 

4. Store amplifiable single-stranded cDNA at -20°C. 

3.2. Synthesis and Immobilization of Single-Stranded Biotinylated 
Homologous Probes to Dynabeads M-280 Streptavidin 

5 '-biotinylated probes may be easily prepared by oligonucleotide synthesis (see 
Note 6) or by PCR. Here, the preparation by PCR from a DNA source containing the 



Homologous Gene Isolation by GC-PCR 367 

gene of interest, i.e.,plasmid or PCR fragment, is described. The design of oligonucle- 
otides used as primer pairs should be performed using a suitable primer design soft- 
ware, i.e., OLIGO. 

3.2. 1. PCR Amplification of Specific cDNA Fragments 
to be Used as Homologous Probes 

Three homologous probes have to be generally designed and synthesized on trans- 
lated exons. The first probe will be designed on terminal exons, the second on middle 
exons, and the third toward the 5' exons. This design will allow the isolation of mol- 
ecules containing the full-length cDNA sequence. For each homologous probe to be 
amplified use 5'-biotinylated forward and nonbiotinylated reverse primer sets: BFP1/ 
RP1, BFP2/RP2, and BFP3/RP3 (see Fig. 1, panel B). 

1. Add the following to the 0.5-mL PCR tube kept on ice: 150 fmoles of DNA template (i.e., 
plasmid, cDNA fragment), 9 |xL 10X Pfu buffer, 3 fxL 10 uM biotinylated forward primer 
(i.e., BFP1, BFP2, and BFP3), 3 uL 10 uM nonbiotinylated reverse primer (RP1, RP2, 
and RP3), 10 uL 2.5 mW dNTP, 5 uL glycerol, and H,0 up to 90 [xL volume. 

2. Add 100 |xL mineral oil and transfer reaction tubes to a thermal cycler. 

3. Denature PCR mixture at 96°C for 5 min then maintain the temperature at 80 C C. 

4. Add 10 u.L of prewarmed Pfu DNA polymerase (2.5 U in IX Pfu buffer) at 80°C 
(see Note 7). 

5. Amplify using the following conditions: 96°C for 30 s, optimal annealing temperature 
(derived from OLIGO software) for 30 s, 75 °C for 3 min, 25 cycles with a final extension 
step of 5 min. 

6. Store at 4°C up to purification. 

7. 5 uL of biotinylated PCR product (BFP1/RP1, BFP2/RP2, and BFP3/RP3) is analyzed by 
agarose gel electrophoresis following standard procedure. 

8. If the expected product is unique, the PCR mixture may be directly ultrafiltered by 
Microcon 100 and four ultrafiltration steps with 500 (xL TEN buffer (500 x g for 12 min) 
(see Note 8). Otherwise, purify the expected fragment by conventional procedure. The 
final volume is adjusted to 100 uL with TEN buffer. 

3.2.2. Immobilization of Biotinylated PCR Product 
(Homologous Probe) to Dynabeads M-280 Streptavidin 

1. For each homologous probe to be prepared add 100 (xL of SV-magnetic beads suspension 
(1 mg) (Dynabeads M-280 Streptavidin 10 mg/mL) in an Eppendorf tube. Use the 
Dynabeads following the Dynal instructions. 

2. Apply the magnet using the magnetic particle concentrator for Eppendorf microtubes 
(Dynal MPC-E). When the liquid phase is completely separated from the solid phase, 
discard the liquid phase. 

3. Add 100 [xL TEN, mix beads, apply the magnet and wait separation, discard the liquid 
phase. 

4. Repeat twice the previous washing step. 

5. The biotinylated PCR product (BFP1/RP1, BFP2/RP2, and BFP3/RP3) is added (90 fxL) 
to the beads, mix beads, and incubated for 30 min at room temperature with gentle agita- 
tion (immobilization step). 

6. Apply the magnet, wait separation and transfer the liquid phase to an Eppendorf tube. 
This liquid phase will be used to check the efficiency of the immobilization step. 



368 Mastrangeli and Donini 

7. Wash the beads containing the immobilized probe three times with 100 uE TEN as 
described in step 3. 

8. Add 100 (xL TEN; mix beads and store at 4°C up to denaturation. 

9. Check the immobilization efficiency by loading on a suitable agarose gel 5 uE of 
biotinylated probe before binding and 5 uE of the supernatant after the binding reaction. 
A decrease of ethidium bromide signal between the pre- and postbinding indicates that 
most of the probe is immobilized. 

3.2.3. Single-Stranded Homologous Probe Immobilized 
to Dynabeads M-280 Streptavidin 

1. Resuspend by mixing the magnetic beads linked to double-stranded homologous probe. 
Apply the magnet and discard the TEN liquid phase. 

2. Add 100 uE of 0.15 M NaOH and incubate for 10 min at room temperature with gentle 
agitation. The double-stranded probe is denatured. 

3. Apply the magnet and discard the liquid phase containing the single-stranded reverse 
sequence not linked to magnetic beads. 

4. Add 100 u.L of 0.15 M NaOH, mix beads, apply the magnet and discard the liquid phase 
(fast wash with denaturing solution). 

5. Wash the beads three times with 100 uL TEN as described in Subheading 3.2.2., step 3. 
The magnetic beads contain now the forward sequence of the single-stranded homolo- 
gous probe. 

6. Add 200 uL TEN and store beads at 4 C C. For long-term storage add 0.02% sodium azide. 

PCR fragments BFP1/RP1, BFP2/RP2, and BFP3/RP3 generate the homologous 
capture probes CP1, CP2, and CP3, respectively (see Fig. 1, panel B). 

3.3. Selection by Capture Steps 

Start the selection procedure using the homologous capture probe CP1 that is 
designed more closely to the 3' end of the encoding DNA sequence. The second cap- 
ture steps will be performed with nonoverlapping homologous capture probe (capture 
probe 2, CP2) that is upstream to CP1. If necessary, a third capture step with a third 
capture probe (CP3) further upstream to CP2 will be performed. 

3.3.1. First Capture Step 

1. In an Eppendorf tube add 50 uE suspension of SV-magnetic beads linked to homologous 
capture probe 1 (CP1), mix beads, apply the magnet, and wait separation, discard the 
liquid phase (see Note 9). 

2. Add 100 uE hybridization buffer (6X SSC, 0.1% SDS), mix beads, apply the magnet, and 
wait separation, discard the liquid phase. 

3. Repeat twice the previous step, then add 100 uL hybridization buffer (prewarmed at 70 C C). 

4. Denature the amplifiable poly (A)-tailed single-stranded cDNA by incubating at 90°C for 
10 min. Chill on ice. 

5. Add 10 u,L of denatured amplifiable poly(A)-tailed single-stranded cDNA (equivalent to 
0.8 ug total RNA) to beads. Mix gently. 

6. Incubate the mixture for 10 min at 70 C C with gentle agitation. 

7. Incubate the hybridization mixture for 4 h in hybridization oven at 45°C under gentle 
rotation to maintain the beads in suspension. 



Homologous Gene Isolation by GC-PCR 369 

8. Mix beads, apply the magnet, and wait separation, discard the liquid phase. Hybrids 
between the homologous capture probe 1 (CP1) and poly(A)-tailed single-stranded cDNA 
are retrieved from the solution by the MPC-E magnet. 

9. Add 100 [xL hybridization buffer, mix beads, apply the magnet, and wait separation, dis- 
card the liquid phase. 

10. Repeat twice. Resuspend in 300 uE of the same buffer. 

11. The beads suspension is divided into three 100 uE aliquots (each containing hybrids deriv- 
ing from 0.27 u.g starting total RNA). 

12. Using prewarmed solutions, wash each aliquot five times as described in step 2 at differ- 
ent temperatures to determine the optimal selection conditions, i.e., 45°C, 55°C, and 65°C, 
respectively (see Note 10). 

13. Equilibrate the beads containing hybridization complexes at room temperature by two 
washing steps with 100 uE of TEN. The suspension can be stored at 4°C up to the next 
elution step (see step 14). 

14. Mix beads, apply the magnet, wait separation, and carefully discard all the liquid phase. 
Resuspend the beads containing hybridization complexes with 100 uE IX TE buffer. 
Incubate at 90 C C for 5 min. The captured poly(A)-tailed single-stranded cDN A molecules 
are released into solution. 

15. Apply the magnet to the warm solution, wait separation, and carefully collect the liquid 
phase, which contains released single-stranded cDNA molecules captured by CP1 homol- 
ogous probe. 

16. Add bovine serum albumin (BSA) (50 u,g/mL final concentration) to the recovered cDNA 
molecule and store at -20 C C. 

3.3.2. PCR of Single-Stranded cDNA Molecules 
Derived from the Capture Step 

1. In a 0.5-mL PCR tube, add 30 uE of amplifiable single-stranded cDNA, 0.5 u,L oligo dT 
mix (10 \iM each primer), 7.5 uL 10X Taq buffer (without MgCl 2 ), 8 uL DMSO, 21 uE 
25 mM MgCl 2 , 8 uE 6.25 mM dNTP. The final volume is 75 uE. 

2. Add 80 uE mineral oil and transfer reaction tubes to a thermal cycler. 

3. Denature PCR mixture at 95°C for 5 min, and then maintain the temperature at 80°C. 

4. Add 5 uE of prewarmed Taq DNA polymerase (2.5 U in IX Taq buffer) at 80°C (see Note 7). 

5. For the first PCR cycle, use the following conditions: 96 C C for 30 s, 48°C for 2 min, and 
72°C for 30 min (see Note 11). 

6. The first cycle is followed by a denaturation step at 96°C 4 min, 80 C C 5 min. 

7. Add 6 uL of 10 \iM AP primer at 80°C (see Note 12). 

8. Amplify for 35 cycles using the following conditions: 96°C for 30 s, 60°C for 30 s, 72°C 
for 4 min. Final extension time 10 min. 

9. Store at 4°C. 

3.3.3. Southern Blot of Captured and Amplified cDNA 

The first capture steps may be easily followed by Southern blot analysis with the 
homologous probe 2 (see Note 13). 

3.3.3.1. Synthesis of the Probe for Southern Blot 

This step provides a DIG-labeled single-stranded probe for the chemiluminescent 
detection of hybridizing molecules. 



370 Mastrangeli and Donini 

1. Add to 0.5 mL PCR tube: 15 ng of DNA template (i.e., PCR fragmens BFP2/RP2, or 
BFP3/RP3 obtained in Subheading 3.2.1.), 20 pmoles of reverse primer (RP2 or RP3), 
9 uL 10X Taq buffer, 6 uL 25 mM MgCl 2 , 10 uL of PCR DIG-labeling dNTP mix, and 
H 2 up to 90 uL volume. 

2. Add 100 u.L mineral oil and transfer the reaction tube to a thermal cycler. 

3. Denature the mixture at 95°C for 5 min then maintain the temperature at 80 C C. 

4. Add 10 uL of prewarmed Taq DNA polymerase (2.5 U in IX Taq buffer) at 80°C. 

5. Perform 25 cycles using the following conditions: 96°C for 30 s, annealing temperature 
(derived from OLIGO software) for 30 s, 72°C for 2 min. 

6. Store the reaction mixture at -20°C until required. 

3.3.3.2. Southern Blot 

1. Ten microliters of amplified cDNA from Subheading 3.3.2., 10 uL of DIG-labeled and 
nonlabeled molecular weight markers are subject to agarose-gel electrophoresis and blot- 
ted onto a positively charged nylon membrane by conventional procedure. 

2. The blot is prehybridized for 60 min at 45 C C with hybridization buffer (0.25 mL/cm 2 filter). 

3. Remove the prehybridization solution and add 0.2 mL/cm 2 filter of hybridization solution 
containing 50 uL of DIG-labeled probe that has been previously treated for 10 min at 
70°C. Incubate the blot overnight at 45°C with gentle agitation. 

4. Remove the filter and rinse with 5X SSC for 2 min at room temperature. 

5. Wash the filter four times with IX SSC, 0.1% SDS (0.7 mL/cm 2 filter) for 30 min with 
agitation at a suitable temperature depending on the requested stringency (see Note 10). 

6. Remove the filter and equilibrate in buffer A for 2 min at room temperature. 

7. Filter is incubated in buffer B (blocking buffer) (0.2 mL/cm 2 filter) for 30 min at room 
temperature. 

8. Remove the blocking solution and add 0.2 mL/cm 2 filter of Anti-Digoxigenin-POD solu- 
tion. The Anti-Digoxigenin-POD solution is prepared by diluting in Buffer B (1:10,000) 
the Anti-Digoxigenin-POD stock solution (150 U/mL in 100 mM Tris-Cl, 150 mM NaCl, 
pH 7.5). Incubate for 30 min at room temperature with gentle agitation. 

9. Remove the filter and rinse with buffer A for 2 min at room temperature. 

10. Wash the blot twice with 0.7mL/cm 2 filter of washing buffer for 15 min at room tempera- 
ture and vigorous agitation. 

11. Remove the filter and rinse with 2X SSC for 2 min at room temperature. 

12. Place the filter on filter paper to drain the liquid, then on a flat container. 

13. Hybrid detection is performed with ECL reagents: mix an equal volume of detection solu- 
tions 1 and 2 and add (0.15 mL/cm 2 ) directly to the blot on the side containing the DNA. 
Incubate for 1 min at room temperature. Drain off excess detection buffer and place the 
filter between two sheets of Photogene development folder or alternatively wrap filter in 
plastic wrap. Gently smooth out air pockets and in a darkroom (red safelight on) place the 
filter in a cassette (DNA side up). Cover with a sheet of Hyperfilm-ECL and expose for 1, 
10, and 60 min, depending on the required exposure time. Develop the film to detect hybrids 
between the probe and the DNA fragments as well as the digoxigenin-labeled MW markers. 

3.3.4. Second Capture Step 

In this step, the magnetic beads linked to the homologous single-stranded probe 2 
(Capture Probe 2, CP2) are used to bind cDNA released from the first capture step 
to select the cDNA molecules sharing homology with both CP1 and CP2 probes (see 
Fig. 1, panel C). 



Homologous Gene Isolation by GC-PCR 371 

1. In a 0.5-mL tube, pool the Southern positive fractions of released single-stranded cDNAs 
from Subheading 3.3.1., step 16, total volume ranges from 70 [xL up to 210 uE. 

2. Incubate at 90°C for 10 min. Chill on ice. 

3. In a 1.5-mL Eppendorf tube, add 138 [xL of the denatured-pooled fractions (if only one 
fraction is available, add 70 uE of this fraction and 68 [xL of 10 mM Tris-HCl, 1 m/W 
EDTA, pH 8.0, BSA 50 [xg/mL instead), 60 uE 20X SSC and 2 uE 10% SDS. Final 
volume is 200 uE. 

4. In a 1.5-mL Eppendorf tube, add 50 uE suspension of magnetic beads linked to homolo- 
gous probe 2 (CP2) (0.25 mg beads), mix beads, apply the magnet, and wait separation; 
discard the liquid phase. 

5. Add 200 |xL hybridization buffer, mix beads, apply the magnet, and wait separation, dis- 
card the liquid phase. 

6. Add 200 uE solution from step 3 to CP2 beads from step 5. 

7. Incubate the mixture for 10 min at 70°C with gentle agitation. 

8. Incubate the hybridization mixture with gentle agitation for 4 h at a single stringent tem- 
perature according to results of the previous Southern blot. 

9. Mix beads, apply the magnet and wait separation, discard the liquid phase. Hybrids 
between the homologous capture probe CP2 and amplifiable single-stranded cDNA are 
retrieved from the solution by the magnet. 

10. Add 200 uE hybridization buffer, mix beads, apply the magnet, and wait separation; dis- 
card the liquid phase. Repeat twice. 

11. The suspension in 200 uE hybridization buffer is divided into two 100 uE aliquots. 

12. Wash each aliquot 5X at the hybridization temperature with either 6X SSC, 0. 1% SDS or 
at more stringent conditions with IX SSC, 0.1% SDS. 

13. Equilibrate the beads containing hybridization complexes by two washing steps with 
100 uE TEN. The suspension can be stored at 4°C up to the next elution step. 

14. Apply the magnet, wait separation, and discard the liquid phase. Resuspend the beads 
containing hybridization complexes with IX TE. Incubates at 90°C for 5 min. 

15. Apply the magnet to the warm solution, wait separation, and carefully collect the liquid 
phase, which contains released single-stranded cDNA molecules selected by CP1 and 
CP2 homologous probes. 

16. Add BSA (50 (xg/mL final concentration) to the recovered cDNA and store at -20°C. 

3.3.5. Southern Blot Analysis After the Second Capture Step 

Thirty microliters of each recovered fraction is amplified by PCR and analyzed by 
Southern blot with a DIG-labelled probe as described in Subheadings 3.3.2. and 3.3.3. 
(Fig. 1, panel D). This analysis should now confirm the isolation of the putative homol- 
ogous gene and indicate whether an additional capture step is required (see Note 14). 

To evaluate by PCR and Southern blotting the capture step efficiency, the controls 
should include 1 . starting amplifiable single-stranded cDNA, 2. cDNA released from the 
first capture step, and 3. cDNA released from the second capture step (see Note 14). 

3.3.6. Third Capture Step 

Perform this additional capture step only if necessary. In this case follow Subhead- 
ing 3.3.4. by using the homologous capture probe 3 (CP3) instead of CP2 on the 
released single-stranded cDNA from the second capture step (Fig. 1, panel C). Spe- 
cific amplified fragments corresponding to the full-length homologous gene should be 



372 Mastrangeli and Donini 

obtained by PCR. A further Southern blot analysis with CP3 probe might be performed 
to get a general view of the applied GC-PCR procedure by adding the necessary con- 
trols and following the steps in Subheadings 3.3.2. and 3.3.3. 
Please see Note 15 if GC-PCR fails. 

3.4. Cloning of Captured cDNA 

The cloning step is necessary for DNA sequencing of the captured DNA (see Note 
16). The user will choose his own preferred cloning and screening strategy. 

It is advisable to use Escherichia coli (E. coli) strain that allows the cloning of 
unstable DNA structures such as inverted repeats, which are present at 5' and 3' ends 
of the obtained fragment (see Note 17). The following are very general instructions: 

1 . Gel purify the selected PCR fragment. 

2. Ligate the PCR fragment to the chosen plasmid vector. 

3. Transform a suitable E. coli strain. 

4. Screen recombinant plasmids. 

5. Select a positive plasmid for DNA sequencing. 

3.5. DNA Sequencing and Analysis 

Plasmid DNA is sequenced to finally confirm the cloned cDNA is the gene of interest. 

3.5.1. DNA Sequencing of Captured cDNA 

1. To start DNA sequencing, use universal primers present on the plasmid vector both 
upstream and downstream to the cloned insert. 

2. Use the most suitable sequencing chemistry for your equipment. 

3. For long DNA fragments, DNA sequence obtained by the universal primers may be used 
to design additional sequencing primers to complete the sequencing of the internal DNA. 
Available primers used to obtain the homologous probes (BFP1, BFP2 BFP3, RP1, RP2, 
and RP3) can also be used as sequencing primers if the homology is very high and the 3' 
end perfectly matches with the homologous gene; the annealing temperature should be 
decreased accordingly as the homology decrease. 

4. Complete DNA sequence on both strands of the captured DNA fragment following the 
most suitable sequencing strategy. 

3.5.2. Confirmation of the DNA Sequence 

Taq DNA polymerase and a modified PCR buffer were used to obtain the prelimi- 
nary sequencing data on isolated fragment (PCR1). These conditions increase the yield 
and also the amplification error rate. Therefore, the obtained sequence has to be con- 
firmed by two additional independent RT-PCR amplifications (PCR2 and PCR3). 

1. Design PCR primers on the extremities of isolated homologous gene (see Note 18). 

2. With the designed primer pairs, perform two RT-PCR reactions on starting RNA (PCR2 or 
PCR3). At least one PCR amplification has to be performed with a different enzyme (with 
increased fidelity, i.e., Pfu) and in selected conditions that minimize misincorporations. 

3. Perform DNA sequencing on both strands of purified PCR2 and PCR3 fragments. 

4. Align the sequencing data of the isolated fragment (PCR1) and those derived from PCR2 
and PCR3. 

5. Determine the correct DNA sequence of the isolated gene. The resulting consensus sequence 
will be unequivocally the correct DNA sequence of the target homologous gene (see Note 19). 



Homologous Gene Isolation by GC-PCR 373 

4. Notes 

1. The 5'RACE system kit (Life Technologies) contains most of the reagents used in this 
procedure, i.e., terminal deoxynucleotide transferase (TdT), Superscript, E. coli RNase 
H, TdT buffer, 0. 1 M DTT and 10 mM dNTP. In addition, if it is preferred to perform a dC 
tailing to facilitate the cloning, the kit contains reagents to perform dC homopolymeric 
tailing and the 5'RACE anchor primer for the further PCR amplification. 

2. The RT reaction is a critical step for long retrotranscripts (12). In our RT-PCR conditions, a 
DNA smear larger than 300 bp is expected. Indeed, a smear between 500 and 2700 bp with 
higher intensity in the 700-1 150 bp region was observed in ethidium bromide stained gel 
(9). The reaction conditions of reverse transcription should favour the synthesis of full- 
length cDNA molecules. Commercial kits are now available for this purpose. 

3. This step is critical and is necessary to remove residual primers, dNTP, degraded RNA 
and single-stranded cDNA molecules shorter than 300 nucleotides. Unwanted TdT reac- 
tions such as different nucleotide incorporation or primer tailing are therefore avoided. 
Store all Microcon 100 filtrates, which in case of leakage should be concentrated and 
filtered with a new device. 

4. Different procedures or commercial kits may be used to obtain amplifiable single-stranded 
cDNA: terminal deoxynucleotide transferase (TdT) tailing (12), single-strand ligation of 
cDNA or "SLIC strategy" (13), Gene Racer kit from Invitrogen, and the Switch Mecha- 
nism at the 5' end of RNA Template (SMART technology) by using the SMART oligo- 
nucleotide during RT reaction (see SMART™ kit, Clontech). 

5. The disadvantage in our method is that the PCR product has to be cloned to obtain the 
DNA sequence. Possible problems with the inverted repeat at the 5' and 3' ends such as 
the suppression PCR effect (14) were not observed (9). 

6. When capture probe sequence is shorter than 100 nucleotides, the 5' biotinylated forward 
homologous probe might be easily synthesized. Biotin may be directly introduced to 5' 
terminus of the capture probes during automated synthesis by using LC biotin-ON from 
Clontech. For a longer sequence, use the described PCR procedure. 

7. Hot-start procedure (15) should be used to prevent mispriming and primer dimerization. 
When adding further reagents, i.e., the prewarmed enzyme, be careful to add the solution 
under the mineral oil layer. 

8. The purification step is necessary to remove the residual biotinylated primer that could 
affect the next binding step to SV-magnetic beads. 

9. From Dynal instructions, 1 mg beads can bind 10-30 pmoles of DNA molecules with 
50-3000 nucleotides in length, at inverted ratio with respect to the DNA length. 0.25 mg 
SV-magnetic beads can bind 2.5-7.5 pmoles of homologous capture probe. For sequences 
100-1000 nucleotides in length, the bounded amount might range from 0.25 to 0.825 jig 
of probe in 100-200 u,L hybridization volume. A high-probe concentration ranging from 
1 .25 to 8.25 u.g/mL is, therefore, obtained allowing fast hybridization reactions compared 
to conventional library screening. 

10. Incubation temperature should be adjusted depending on the required stringency. The salt 
concentration may also be decreased to increase the stringency. OLIGO program pro- 
vides melting temperatures at several salt concentrations for a given probe and for vari- 
ous homology conditions. As the homology is not a priori known, the first step is 
performed in conditions that allow hybridization of molecules sharing a homology higher 
than a minimal assumed value, i.e., 60%-70%. 

11. PCR must be performed in conditions suitable to obtain full-length molecules, especially 
in the critical first cycle when amplification fails if the full-length double stranded cDNA 



374 Mastrangeli and Donini 

is not obtained. Captured cDNA is amplified by PCR with Taq DNA polymerase accord- 
ing to the method of Frohman (12), with minor modifications. A buffer composition 
(6.5 mM MgCl 2 , 10% DMSO, 1.5 mM dNTP) was found by Frohman to give high ampli- 
fication yield of cDNA. 

12. AP primer sequence is present on 5' terminus of both forward and reverse sequence. If a 
different tailing is performed two different anchor primers have to be used. Anchor 
sequences containing suitable cloning site might also be designed to facilitate the down- 
stream cloning step. 

13. Although Southern blot after the first capture step may be omitted to speed-up the proce- 
dure, it provides useful information on both CPl-captured cDNA and CP2 capture probe. 
The ethidium bromide stained gel shows the complexity of CPl-captured material at vari- 
ous washing temperatures. The Southern positive signal with the homologous probe 2 
used at a given washing stringency will give indications on the presence of the target 
gene, its expected size and specificity when compared to ethidium bromide signals. Fur- 
thermore, it will give indications that the second homologous probe may be successfully 
hybridized and washed at a given temperature in the further capture step. The use of a 
probe corresponding to the sequence of the first capture probe has to be avoided as it will 
not be specific and almost all ethidium bromide signals will be positive. This is a serious 
problem in the first capture step. 

14. In our study, the homology of capture probes 1 and 2 (713 and 290 nt long, respectively) 
with the gene of interest was found to be 79% and 82%, respectively. After the first and 
nonselective capture step on activated and nonactivated thymocytes, a smear was still 
observed by ethidium bromide staining, but the homologous gene was clearly visible by 
Southern blotting in activated thymocytes only. After the second capture step, ethidium 
bromide stained gel clearly showed only the bands of interest (9). 

15. GC-PCR may fail when: 1. the target gene is present in the mRNA source at a very low 
level (use an activated cell source and/or increase the PCR cycle number); 2. starting 
mRNA is degraded (use a new RNA preparation and avoid RNase contamination); 3. 
homopolymeric tailing fails or tailed cDNA is lost in the Microcon purification step 
(check ethidium bromide staining of the amplified noncaptured cDNA mixture, the 
absence of an intense and smeared signal indicates that cDNA is not amplifiable; repeat 
the tailing reaction with new reagents); 4. first strand cDNA synthesis of long mRNA 
molecules is uncompleted (shorter fragment than expected are observed by ethidium bro- 
mide stained gel after the second capture step, if the 5' encoding region is not present, 
Southern blot will be negative by using a probe corresponding to CP3 region and positive 
with a probe corresponding to CP2. If this is the case, isolation of the fragment followed 
by 5'RACE will provide the full-length gene. Alternatively, design additional capture 
probe closest to 3'encoding sequence or optimise the RT reaction to obtain longer 
retrotranscripts.); 5. Second-strand cDNA synthesis of long cDNA is uncompleted in the 
first PCR cycle (increase extension time, use conditions and enzymes which allow long 
distance PCR). 

16. A different TdT tailing should avoid the cloning step and a direct sequencing of the iso- 
lated fragment may be performed with the anchor primers if an acceptable homogeneity 
of the PCR product is achieved (see Note 1). 

17. SURE strains are available from Stratagene which allow the cloning of unstable DNA 
structures. 

18. Anchored specific primers containing suitable cloning sites might be used to facilitate the 
gene cloning into expression vector for recombinant protein production. 



Homologous Gene Isolation by GC-PCR 375 

19. The sequencing data are derived from three independent RT-PCR reactions, one 
performed with a different enzyme chosen between high fidelity proofreading enzymes 
such as Pfu, ThermalAce (Invitrogen), Pwo (Roche), etc. If present in a given position, an 
error will be present only in one sequence whereas the other two sequences will show the 
correct nucleotide. 

In PCR1 conditions used to maximize the yield (12), several misincorporations were 
observed (9). Two additional PCRs were performed with Tag and Pfu DNA polymerase, 
respectively. Both PCR2 and PCR3 sequences were found identical, suggesting identity 
with the natural sequence (9). 

Acknowledgments 

The authors would like to thank Carlo Serafini for supporting this work and Emilia 
Micangeli for the expert and valuable technical assistance. 

References 

1. Sambrook, J., Fritsh, E. F., Maniatis, T., and Irwin, N. (1989) Costruction and analysis of 
cDNA libraries, Preparation of radiolabeled DNA and RNA probes, Synthetic oligonucle- 
otides probes. Screening expression libraries with antibodies and oligonucleotides, in 
Molecular Cloning, A Laboratory Manual, 2nd edition, (Nolan, C. ed.), Book 2. Chapters 
8, 10, 11, and 12. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 

2. Goffeau, A., Barrell, B. G., Bussey, H., Davis, R. W., Dujon, B., Feldmann, H., et al. 
(1996) Life with 6000 genes. Science 274, 563-567. 

3. The C. elegans Sequencing Consortium. (1998) Genome sequence of the nematode C. 
elegans: a platform for investigating biology. Science 282, 2012-2018. 

4. Adams, M. D., et al. (2000) The genome sequence of Drosophila melanogaster. Science 
287,2185-2195. 

5. The Arabidopsis Genome Initiative. (2000) Analysis of the genome sequence of the flow- 
ering plant Arabidopsis thaliana. Nature 408, 796-815. 

6. Venter, J.C., et al. (2001) The Sequence of the Human Genome. Science 291, 1304-1351. 

7. Pennisi, E. (2000) Genomics. Rat genome off to an early start. Science 289, 1267-1269. 

8. Gibbons, A. (2000) Genomics. Building a case for sequencing the chimp. Science 289, 1267. 

9. Mastrangeli, R., Micangeli, E., and Donini, S. (1996) Cloning of murine LAG-3 by mag- 
netic bead bound homologous probes and PCR (gene-capture PCR). Analyt. Biochem. 
241,93-102. 

10. Liang, P., Zhu, W., Zhang, X., Guo, Z.,. O'Connell, R. P., Averboukh, L., Wang, F., and 
Pardee, A. B. (1994). Differential display using one-base anchored oligo-dT primers. Nucl. 
Acids Res. 22, 5763-5764. 

11. Mastrangeli, R. and Donini, S. (1998) Gene-Capture PCR in BioTechniques Books: Gene 
Cloning and Analysis by RT-PCR, (Siebert, P.D., and Larrick, J. eds.), Eaton, Natick, MA. 
pp 271-288. 

12. Frohman, M. A. (1993) Rapid amplification of complementary DNA ends for generation 
of full-length complementary DNAs: thermal RACE. Meth. Enzymol. 218, 340-356. 

13. Edwards, J. B., Delort, J., and Mallet, J. (1991) Oligodeoxyribonucleotide ligation to 
single-stranded cDNAs: a new tool for cloning 5' ends of mRNAs and for constructing 
cDNA libraries by in vitro amplification. Nucl. Acids Res. 19, 5227-5232. 

14. Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., et al. 
(1996) Suppression subtractive hybridization: a method for generating differentially 



376 Mastrangeli and Donini 

regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93, 
6025-6030. 
15. Chou, Q., Russell, M., Birch, D. E., Raymond, J., and Bloch, W. (1992) Prevention of pre- 
PCR mis-priming and primer dimerization improves low-copy-number amplifications. 
Nucl. Acids Res. 20, 1717-1723. 



39 

Rapid and Nonradioactive Screening 
of Recombinant Libraries by PCR 

Michael W. King 
1. Introduction 

The advent of the polymerase chain reaction (PCR) has greatly facilitated the 
isolation and characterization of clones from both cDNA and genomic libraries (1-3). 
Given the complexity of the genome of a particular organism or the relative abun- 
dance of a particular mRNA, within the cell type from which a cDNA library was 
constructed, the ability with which one can isolate a gene or cDNA clone of interest is 
quite variable. With respect to genomic libraries, the number of clones needed to be 
screened to isolate a single-copy sequence is a function of the complexity of the 
genome and the average size of the cloned fragments in the library. In the case of 
cDNA libraries, the frequency of a given clone of interest depends on the abundance 
of the messenger RNA. Highly abundant messages can represent 10% or more of total 
mRNA, whereas, very rare messages can be as low as one in 10 6 . In addition, the 
representation of some sequences in a cDNA library, particularly the 5' ends of large 
mRNAs, will be less than expected owing to the technical difficulties in converting 
the mRNA into full-length cDNA copies. In some cases, a particular sequence of 
interest can be depleted or lost at various steps of screening owing to its inefficiency 
to be replicated relative to other clones in the library. 

Prior to the advent of the PCR, the principal technique for screening bacteriophage 
X-based libraries involved screening nitrocellulose filters replicas with radioactively 
labeled probes. Recently, more highly sensitive methods for screening that utilize the 
PCR have been described (1-3). This chapter describes a PCR-based selection method 
for the isolation of clones from recombinant DNA libraries prepared in X-based 
vectors. The technique uses no radioisotopes and can be completed in as few as 7 d. 
The technique is amenable to the use of highly specific PCR primers as well as degen- 
erate primers designed to isolate families of related clones. In this method, a cDNA or 
genomic library is initially plated on as few as ten 100 mm plates at a density near 
3000-5000 plaques per plate. The phage from each plate are soaked in SM buffer (4) 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

377 



378 King 

to generate the starting aliquots for PCR. Positive aliquots from the primary plating 
are identified and replated at lower and lower densities to generate subaliquots for 
secondary and tertiary screens until a positive clone is identified by PCR of phage 
soaked from a single plaque. 

PCR-based library screening can be performed on both cDNA libraries and genomic 
libraries that are cloned in any variation of bacteriophage X-based vectors. Screening 
can be performed from aliquots of unamplified newly packaged libraries or from plate 
lysate amplified libraries. It is helpful, yet not necessary, to know the titer (plaque 
forming units per milliliter; pfu/mL) of the library being screened. In general, good 
primary libraries have a titer of anywhere from 1,000,000-5,000,000 pfu in the entire 
initial packaging reaction (usually a 500-ixL library). Libraries that have been 
amplified generally have a titer of 10 10 -10' 'pfu/mL. Unless it is known that the titer of 
a given library is uncharacteristically low, determining the precise titer of the starting 
aliquot is not necessary (see Subheading 3.5.). 

2. Materials 

1. Recombinant DNA library. 

2. Oligonucleotide primers for PCR (see Subheading 3.1.). 

3. Bacterial culture media, agar plates and top agar media. 

4. SM Buffer (4): 100 inM NaCl, 8 mM MgS0 4 , 50 mM Tris-HCl, pH 7.5. 

5. Reagents for PCR. 

6. Reagents for agarose gel electrophoresis. 

7. CHC1 3 . 

3. Methods 

3.1. Design of Primers 

1. In designing primers for PCR one needs to consider several important factors such as 
making the primers with a near 50% GC content, a high degree of specificity with respect 
to nucleotide sequences and the absence of primer self-complementarity. The 
GenomeWeb site (http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html) main- 
tained by the UK. HGMP Resource Center has links for several sites supplying primer 
design software tools to aid in the proper design of primers for PCR-based techniques. 
Primers that are from 18-21 nucleotides in length are optimal for this (and most) PCR- 
based technique because they allow high annealing temperatures that result in greater 
specificity in the reaction. In most cases, where possible, the primers should be designed 
to be maximally useful in a PCR with 2 mM MgCl 2 and an annealing temperature above 
62 C C. It has been shown that primer annealing is very specific over a wide range of MgCl 2 
concentrations (from 0.5 mM to 2 mM). In addition, a higher degree of fidelity can be 
obtained, in some cases, by substitution of MgS0 4 for MgCl 2 . 

2. With the use of degenerate primer pairs, as for the isolation of families of related cDN As 
or genes, it is extremely important that the 3'-nucleotide position of each primer set be 
nondegenerate in order to prevent an increase in nonspecific templating. In most cases, it 
is also optimal if the annealing temperature used with degenerate primers is higher than 
55°C, although examples are available where successful isolation of related cDNAs has 
been carried out using degenerate primer pairs with annealing temperatures of 52°C. 



Screening of Libraries by PCR 379 

3. For increased specificity it is possible to add a nested primer that resides within the 
sequences to be amplified with the primary primer pair. This nested primer can then be used 
in a second PCR, in combination with one of the primary primers, to test positive PCRs 
for the presence of correct internal sequences. 

3.2. Basics of Phage Growth 

1. Each step in this PCR-based screening protocol begins with the overnight culture of the 
appropriate host Echerichia coli (E. coli) (see Table 1). The cells should be grown at 
37°C with agitation in NZCYM media supplemented with 1 mM MgS0 4 and 0.3% mal- 
tose to ensure optimal phage infection and growth (4). The minimum volume of the over- 
night culture of cells depends upon the number of plates that will be used. The standard 
volume of cells is 100 (xL of bacteria for each 100 mm plate. 

2. The next morning an aliquot of top agar is melted and held at 45°C. The appropriate 
number of agar plates are prewarmed at 37°C for 15-20 min. 

3. The library or plate lysate aliquots are diluted appropriately in SM buffer, then added to 
an aliquot of the fresh overnight host E. coli that corresponds to the total volume needed 
for plating. Adding the phage to the entire volume of cells, instead of to individual 
aliquots, ensures an equal distribution of phage on each plate. The cells and phage are 
then incubated at 37°C for 10 min to allow the infection cycle to initiate. 

4. The infected cells are then separated into individual aliquots for plating. The plating is 
carried out by adding melted 45 C C top agar to each tube (3 mL for each 100 mm plate), 
pouring the solution onto individual plates and ensuring even spreading of the top agar. 

5. Let the plates sit for 10 min at room temperature to harden the top agar. The plates are 
then inverted and incubated at 37°C for 6-8 h to allow plaques to form. (See Subhead- 
ings 3.5.-3.8. for the screening protocol.) 

3.3. Preparation for Screening 

1 . It is critically important that, prior to screening any library with this technique, the library 
be tested for the presence of clones that contain sequences that will amplify with a given 
primer pair. 

2. Test a l-u,L aliquot (undiluted) of the library to be screened using the standard PCR 
protocol described in Subheading 3.4. As described in the Introduction, an amplified 
library will have approx 10 7 — 10 K phage in a 1-u.L aliquot. Therefore, a 1-jiL aliquot will 
contain 100- to 1000-fold more phage than is statistically necessary to screen to find a 
clone of a given sequence. If the primers are unable to amplify the correct fragment from 
this amount of phage then the library is either devoid of clones or the primer pair is not 
functional as expected. 

3.4. Standard PCR 

1. All PCRs are performed in a volume of 25 u.L containing IX polymerase buffer, 2 mM 
MgCl 2 (or a concentration appropriate for a given primer pair), 10 pmole of each primer 
(approx 60 ng; assuming primers of 18-21 nucleotides) and 200 \xM deoxynucleotide 
5'-triphosphates (dNTPs). It is optimal to prepare a master mix (of sufficient volume for 
all the reactions) containing all components of the PCR except the template. This ensures 
equivalence of components in each individual reaction. It is important to note that an 
excess of any component of the PCR, in particular, an excess of primers, can lead to 
failure of the reaction or false positive results. 



380 



King 



Table 1 

Outline of Screening Protocol 



Day 



Activity 



Identity 



1 Start culture of host E. coli 

2 Plate 5-10 100-mm plates (approx 3000-5000 pfu/plate), Primary screen 
grow 6-8 h, soak phage in SM overnight at 4°C 

3 Process phage lysates, PCR screen Primary screen 

3 Start culture of host E. coli 

4 Plate 5 100 mm plates, of 1 or all 1° positives (approx Secondary screen 
1000 pfu/plate), grow 6-8 h, soak phage 

in SM overnight at 4°C 

5 Process phage lysates, PCR screen Secondary screen 

5 Start culture of host E. coli 

6 Plate 5 100-mm plates, (approx 100-500 pfu/plate), Tertiary screen 
grow 6-8 h, soak phage in SM overnight at 4 C C, is 

possible to pick single plaques at this stage 

7 Process phage lysates, PCR screen Tertiary screen 

7 Start culture of host E. coli 

8 Plate 1 100 mm plate (approx 50 pfu/plate), grow 6-8 h, Final screen 
pick single plaques into SM and elute overnight at 4°C 

9 Screen single plaque lysates by PCR Final screen 



2. To ensure that the phage particles in the plate lysates are disrupted, the PCRs are "hot 
started." This is accomplished by an initial denaturation at 95°C for 10 min. The reaction 
should then be held at 80°C whereaas adding polymerase. Many vendors sell Taq poly- 
merase prebound by antibody. This prevents the enzyme from acting on any template 
until the antibody is denatured by the "hot start" conditions. Using these forms of poly- 
merase, it is possible to add the enzyme directly to the master mix. 

3. The following cycle profiles have been shown to be optimal for the majority of specific 
primers (see Notes for degenerate primers): 

30 cycles: 95°C, 0.5-1 min (denaturation); n°C, 0.5-2 min (annealing temperature 
defined by primer sequences, see Note 1); 72°C, n min (extension time depends on prod- 
uct length, for most reactions 0.5 min is sufficient; see Note 2). 

3.5. Primary Screen 

1. For the primary screen it is usually necessary to plate 10 aliquots of the library at a density 
in the range of 3000-5000 pfu/plate. This density will nearly destroy the entire bacterial 
lawn. If the titer of the library is unknown, most amplified libraries can be plated using 
1 u.L of a 10 3 dilution dispersed onto the 10 plates (i.e., the equivalent of 0.1 u.L/plate). In 
some cases, it is possible to screen as few as 5 plates. 

2. Incubate the plates upside down at 37°C. Stop the incubation when the plaques begin to 
merge with one another. This usually takes 6-8 h at 37°C. 



Screening of Libraries by PCR 38 1 

3. Overlay the plate with 4 mL of SM buffer and let stand at 4°C overnight. It is possible to 
incubate the plates with SM buffer for 2 h at 37°C or room temperature for 4-5 h. How- 
ever, the PCR results are frequently smeary because of bacterial growth in the SM buffer. 
Also, the titer of the resultant lysates can be 100- to 1000-fold lower than the lysates 
prepared by 4 C C overnight incubation. This latter fact is important to remember for sub- 
sequent screens. 

4. Collect the SM buffer as a separate aliquot from each of the plates and remove agar and 
bacterial debris by centrifugation at 3000g for 10 min in a JA17 rotor (Beckman high- 
speed centrifuge) or an SS34 rotor (Sorval centrifuge). 

5. Save 1 mL from each aliquot. Add CHC1 3 to 0.3% to prevent bacterial growth in these 
aliquots and allow for their longer term storage at 4 C C. 

6. Use a 1-uL aliquot for the PCR assay (see Subheading 3.4.). 

7. Analyze a 15 — (1 aliquot of each PCR on an agarose gel to determine which aliquot(s) have 
amplified the target of interest (see Note 3). 

8. In most screens, there should be at least 1 plate lysate exhibiting a positive signal by PCR 
(see Fig. 1). 

9. Provided the library was tested for the presence of DNA that can be amplified with the 
primer pair being used (see Subheading 3.3.), it will be possible to find primary lysate 
that contain positive signals. It may be necessary to continue to screen more primary 
plates until a positive is found. 

3.6. Secondary Screen (see Note 4) 

1. It is not necessary to titer the primary plate lysates although this can be done if accurate 
plaque numbers in the secondary screen are desired. However, based upon the fact that 
the primary plates should have experienced near complete lysis of the bacterial lawn, the 
secondary screens (using five 100 mm plates) are plated using 1 uL of a 10 4 dilution (i.e., 
0.2 [xL/plate) of each positive primary lysate. Plating at this density (approx 500-1000 
plaques/plate) is to ensure that the secondary lysates are dense enough to allow for 
enrichment of the clones of interest. 

2. Allow plaques to grow as for the primary screen. 

3. Prepare the phage lysates as for the primary screen using 3 mL of SM buffer. 

4. Process the plate lysates and save a 1-mL aliquot of each with 0.3% CHC1 3 for long-term 
storage. 

5. Screen a l-(xL aliquot of each in a 25-jaL PCR as for the primary screen. 

6. Analyze a 10-uL aliquot of each PCR by agarose gel electrophoresis (see Fig. 1). 

3.7. Tertiary Screen (see Note 5) 

1. The tertiary screen is the last plate lysate screen. It is possible, in some instances, to 
proceed directly to the screening of single plaques from the secondary screen. In some 
screens it may be necessary to perform an additional quaternary plate lysate screen prior 
to assay of single plaques (see Notes 6 and 7). 

2. Plate 5 plates (100 mm plates) using 1 u.L of a 10 4 dilution of a positive secondary lysate 
(i.e., 0.2 (xL/plate). In some cases it may be necessary to use a 10 5 dilution, depending 
upon the density of plaques in the secondary screen. 

3. Allow plaques to grow as for the primary screen. 

4. Prepare the phage lysates as for the primary screen using 3 mL of SM buffer. 

5. Process the plate lysates and save a 1-mL aliquot of each with 0.3% CHC1 3 for long-term 
storage. 



382 



King 



Primary Screen 

KT 1 M 4 S & 7 a n in 



Secondary Screen 
HT 1 2 3 * 





Single Plaque Screen 



MT 1 2 3 4 1 ff 



I t M 11 it 1 J 1' 1* 



Tertiary Screen 
mil) 4 s 





Fig. 1. Typical PCR-based library screen results. Ten plate lysates were assayed for the 
primary screen. Lysate #3 was used for the secondary screen. Lysate #3 from the secondary 
screen was used for the tertiary screen. Lysate #3 of the tertiary screen was used for the screen- 
ing of single plaques, a total of 15 were assayed. A single plaque was found to be positive and 
used for phagemid rescue. 



6. Screen a 1-^L aliquot of each in a 25-\iL PCR as for the primary screen. 

7. Analyze a 10-u.L aliquot of each PCR by agarose gel electrophoresis (see Fig. 1). 

3.8. Single Plaque Screen 

1. Plate a single 100 mm plate from a tertiary (or a secondary) positive using 1 \iL of 10 3 or 
10 4 dilution. 

2. The level of dilution used for the single plaque screening plate is less than for a tertiary 
screen and the amount of the dilution plated is 1 u,L. This is because the density of plaques 
in the tertiary screen should have been in the range of less than 250-500 pfu/plate such 
that the lysates from those plates will have a lower titer. 

3. Pick individual plaques into 75-100 (xL of SM buffer using sterile glass Pasteur pipets to 
"scoop" the plaques out of the top agar. The volume of SM buffer used to soak single 
plaques depends on the size of the plaques themselves. Generally, if the plaques are simi- 
lar in diameter to the diameter of a glass Pasteur pipet then one should use 100 fiL of SM 
buffer, less for smaller plaques. 

4. Elute the phage particle from the plaque overnight at 4°C (see Notes 8 and 9). 

5. At this step it is best to screen a 3-u,L aliquot in the standard 25 u.L PCR. Volumes less 
than 3 \iL can be used but have a tendency to give variable amplification (see Fig. 1). 



Screening of Libraries by PCR 383 

4. Notes 

1. The annealing temperature (T m ) used during the PCR, for any given primer, is determined 
from the base composition of the primer. To calculate the annealing temperature use the 
following formula: T m = 2(A+T) + 4(G+C). As an example for a 21mer primer with 12 
G+C and 9 A+T the T m = 2(9) + 4(12) = 66°C. For the majority of PCRs it is optimal to 
use an annealing temperature that is 2-4°C below the calculated T m . However, increased 
specificity is obtained by annealing at the T m . 

2. The elongation time used in the PCR is determined by the length of the resultant product. 
Given the rate of the majority of DNA polymerases at approx 1000 bases per second it 
would, in theory, be possible to use extremely short elongation times. However, in practice 
it has been observed that an elongation time of approx 1 min per 1000 bases is optimal. 

3. To analyze PCR products by agarose gel electrophoresis one should use different per- 
centages of agarose (dependent upon product size) in order to obtain good resolution of 
products. In general use 1.5-2% gels for products of less than 500 bp, 1-1.5% for prod- 
ucts of 500-1000 bp and 0.7-1% for products greater than 1000 bp. 

4. Following the primary screen, each positive plate lysate will most probably represent a 
different type of cDNA or gene clone unless degenerate primers were utilized. Therefore, 
to maximize the possibility of obtaining full-length cDNA clones or overlapping genomic 
clones each primary positive should be carried through to the secondary stage. However, 
to reduce the screening "load" one single primary positive at a time can be carried through 
to single plaque isolation. 

5. When using specific primers, each positive secondary lysate represents the same single 
type of clone that was present in the primary lysate. Therefore, only one of the positive, 
secondary lysates is carried through to the tertiary screen. Also, only a single tertiary 
positive is carried through to the single plaque screen. 

6. When using degenerate primer pairs, the complexity of possible clone types in any given 
primary positive can be large. For this reason it is necessary to plate at least 10 plates for 
the secondary screens. Because of this complexity of clones in a primary positive, it is 
best to carry only one primary positive at a time through to single plaque isolations. In 
addition, because each of the secondary positives will likely represent different types of 
clones, each of them needs to be screened in the tertiary screen. The latter fact is also 
likely in many cases at the level of the tertiary (and beyond) screens. 

7. The use of degenerate primers in this screening technique will require at least four and 
possibly as many as six rounds of plate lysate screening prior to the screening of single 
plaques. 

8. The single plaque lysate is used to prepare phage DNA as well as a permanent stock of the 
clone by small scale liquid lysis. Start an overnight culture of the appropriate host E. coll 
in NZCYM plus maltose and MgS0 4 (see Subheading 3.2.). The next morning add 25 |xL 
of the 100 (xL of SM (into which the positive plaque was eluted) to 100 (iL of overnight 
cells and 100 uL NZCYM with maltose and MgS0 4 . Incubate with agitation at 37°C for 
10 min. Transfer this culture to 50 mL of NZCYM without maltose or MgS0 4 . Incubate 
with agitation at 37°C until the cells in the culture begin to lyse. This takes approx 6 h and 
is visible as debris in the normally silky appearance of the growing E. coli. At this time 
add CHC1 3 to 0.5% and incubate an additional 10 min to accelerate the cell lysis as well 
as to prevent further growth of the cells. Centrifuge the cells and debris at 7K rpm 
for 10 min. Save an aliquot of the supernatant as a stock of the clone, either at 4°C or by 
adding dimethyl sulfoxide (DMSO) to 7% and storing at -80 C C. To the remainder of the 
phage supernatant add RNase A and Dnase I to 1.5 u.g/mL and incubate at 37°C for 



384 King 

30 min. Precipitate the phage particles by addition of solid PEG 6000 to 10% (w/v) and 
solid NaCl to 0.5 M. Place at 4°C overnight. Collect the precipitate by centrifugation at 
9k rpm for 25 min. Resuspend the precipitate in 0.5-1 mL of TE buffer (4). Add Protein- 
ase K to 150 [Ag/mL and incubate at 45 °C for 45 min. Extract the released phage DNA 
with an equal volume of phenol then phenol/CHCl 3 and again with CHC1 3 . Precipitate the 
DNA by addition of 0.1 vol of 2 M ammonium acetate pH 5 and 2 vol of ethanol. The 
DNA should form a stringy precipitate immediately. It is best to remove the precipitat- 
ing DNA by collecting it on a swirling glass rod. This reduced RNA and protein contami- 
nation that may affect restriction enzyme digestion. Centrifuge the precipitate at top speed 
in a microfuge, remove any supernatant and resuspend the pellet in 50-100 uL of TER 
buffer (4). Use from 3-10 \iL for restriction enzyme digestion. 
9. The single plaque lysate is used for the rescue of phagemid DNA, containing the cDNA 
clone, from XZap® (Stratagene, Inc.) or (ZipLox® (Life Technologies, Inc.) if the cDNA 
library was constructed with these modified forms of bacteriophage k. The protocol for 
phagemid rescue from XZap is described. Start a culture of XL 1 -Blue® overnight in 
NZCYM plus 0.3% maltose and 1 mM MgS0 2 . Also start a culture of SOLR® in LB 
media (4). The next morning dilute 40 uL of the overnight XL 1 -Blue cells into 1 mL of 
NZCYM plus 0.3% maltose and 1 niM MgS0 4 . Incubate with agitation at 37°C for 
60 min. Transfer 200 uL of these cells to a new tube, add 25 uL of the 100 [xL SM (into 
which the positive plaque was eluted) and 1 jaL of ExAssist® helper phage. Incubate with 
agitation at 37°C for 15 min. Add 3 mL of LB media (4) and incubate with agitation at 
37°C for 2.5 h. Centrifuge the solution at 2000 rpm for 15 min. Transfer the supernatant 
to a new tube and heat at 70°C for 15 min. Centrifuge at 6000g for 15 min. Save the 
supernatant in a sterile tube as this is a stock of the rescued single-stranded phagemid. 
This solution can be stored at 4°C for up to 2 mo. To obtain colonies with the double- 
stranded phagemid, add 10-50 uL of the phagemid stock solution to 100 uL of the fresh 
overnight SOLR cells. Generally, 10 [xL of the phagemid stock is more than enough to 
yield several hundred colonies when passaged through SOLR. Incubate with agitation at 
37°C for 15 min. Spread 10-50 \xL onto a single LB plus ampicillin plate and incubate 
overnight at 37°C. In most cases a single 10 uL aliquot is sufficient for plating since only 
a single colony is necessary. The double-stranded phagemid DNA can then be isolated 
from colonies by standard miniprep techniques (4). 

References 

1. King, M. W. (2000) Screening recombinant libraries by polymerase chain reaction, in 
PCR Cloning Protocols, in the series: Methods in Molecular Biology, 2nd ed., Humana 
Press, Totowa, NJ. 

2. Amaravadi, L. and King, M. W. (1994) A rapid and efficient, non-radioactive method for 
screening recombinant DNA libraries. BioTechniques 16, 98-103. 

3. Isola, N. R., Harn, H. J., and Cooper, D. L. (1991) Screening recombinant DNA libraries: 
A rapid and efficient method for isolating cDNA clones utilizing the PCR. BioTechniques 
11, 580-582. 

4. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1992) Molecular Cloning: A Laboratory 
Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 443. 



40 

Rapid cDNA Cloning by PCR Screening (RC-PCR) 

Toru Takumi 

1. Introduction 

Now that the draft sequence of the human genome is available (1 ,2), cDNA cloning 
based on its sequence from a library is no longer an experimental goal, but a starting 
point and a routine laboratory practice. Hybridization screening with either radio- 
labeled or nonradio-labeled probes had been commonly used for cDNA cloning before, 
but it is laborious and time-consuming in the post-genome era. Application of the 
polymerase chain reaction (PCR) is surprisingly widespread since its discovery (3). 
Here an application to cDNA library screening is reported: rapid cloning of full-length 
cDNAs by screening pools of cDNAs by PCR (RC-PCR) (4). This PCR-based cDNA 
screening technique is applicable to both bacteria and phage libraries. 

Providing that one has identified unique DNA sequences not only in a PCR clone or 
a partial-length cDNA clone, but also in ESTs or genomic sequences in databases, this 
RC-PCR screening technique is very simple and extremely rapid for isolation of a full- 
length cDNA from a library. This RC-PCR technique involves no radioisotopes, and 
avoids labor-intensive and expensive procedures such as transferring bacterial colo- 
nies to filters and hybridizing them to radiolabeled DNA probes. RC-PCR enables us 
to isolate a single clone from the library in a period of a few days. 

2. Materials 

1. cDNA library (see Note 1). 

2. Reagents for PCR (store at -20°C): 

a. PCR buffer: 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 . 

b. dNTP mixture. 

c. Taq DNA polymerase. 

d. Sterile water. 

3. Sequence specific primers. 

4. 96-well dishes. 

5. LB medium. 

6. Agarose gel for analysis of PCR products. 

From: Methods in Molecular Biology, Vol. 192: PCR Cloning Protocols, 2nd Edition 
Edited by: B.-Y. Chen and H. W. Janes © Humana Press Inc., Totowa, NJ 

385 



386 Takumi 

3. Methods 

3. 1. Primer Design 

1 . The sequence of primers should be specific for the target sequence. In the case of cloning 
members of a gene family, choose the region that is less conserved among the family. 

2. The parameters for designing primers are the length (basepairs), the GC/AT ratio and 
melting temperature (5). 

3. As to the length of primers, a 17-18-mer is thought to be sufficient for RC-PCR if a 
positive control gives a single, specific band in an agarose gel. Longer primers are appro- 
priate to amplify larger PCR products. 

4. Amplification of 200-400 bp DNA is the most efficient (5). 

3.2. PCR Screening 

1. Divide the cDNA library into ten fractions, each of which contains, for example, approx 
100,000 clones (see Note 1). Spread each of the library over 150 mm LB agar plates 
containing the appropriate antibiotics. After overnight growth, collect the colonies by a 
scraper and put them into microfuge tubes containing LB solution. 

2. For each of the ten fractions, assemble a 50-[xL PCR in a 500-uL microfuge tube contain- 
ing the following: 

10X PCR buffer 5.0 uL 

dNTP mix 200 \xM each 

Primer 0.15 \iM each 

DNA template 2.5 (xL of broth containing the colonies (see Notes 2-4) 

sterile H 2 to 49.5 uL final volume 

Taq DNA polymerase 0.5 \iL (2.5 u) 

3. After a 5-min denaturation at 94°C, the cycling parameters are the following (see Note 4): 
25 cycles 94°C 45 s 

60-65 °C 1 min 

72°C 2 min 

4. Run an agarose gel to detect the positive fractions. 

5. For the second screening, dilute the positive pool of cells in LB broth to a concentration 
of about 30,000 clones per mL and distribute 100 (xL of this suspension (about 3000 
clones) into each of the 96 wells of a microplate. Combine 10 uL from each well in a 
column (giving a total of 120 uL from each of the plate's 12 columns); similarly 10 uL 
from each well in a row (giving 80 [iL from each of the 8 rows). From each of the result- 
ing 20 mixtures, directly use 2.5 \iL as the DNA template of the next PCR. 

6. Perform PCR as described in steps 2 and 3 and analyze the results by gel electrophoresis. 

7. Repeat screening and subdividing of the positive pools until a single clone is obtained 
(see Note 5). 

4. Notes 

1. As described earlier, this protocol is applicable to both bacteria and phage libraries. In the 
case of screening of phage libraries, a high phage titer is necessary for RC-PCR in the first 
screening. Once the titer of the positive pools drops to less than the order of 10 3 in a serial 
screening, it is recommended to amplify the phages on a small plate (10 cm) before pro- 
ceeding. For other protocols of the screening of the phage libraries by PCR, see refs. (6-8). 

2. Extraction of plasmid DNA is not necessary for PCR; intact bacterial colonies are suffi- 
cient for use as PCR templates (9,10). LB solution containing bacterial colonies can be 



Screening cDNA Pools by RC-PCR 387 

used directly as a template without any culture. In theory, it is not necessary to incubate 
the medium if it includes a positive clone. Incubation of the broth, however, may help to 
detect the positive signal especially for the initial screening. The number of colonies may 
determine whether the medium should be incubated or not. 

3. The quality of cDNA library determines whether or not it is possible to isolate a full- 
length cDNA or a partial one. Use of primers that correspond to the relatively upper 
stream region of the cDNA may help increase the chances of isolating a full clone. 

4. The PCR conditions should be optimized in each case by use of a positive control. Primer 
design, concentration of magnesium (Mg + ) and annealing conditions, including tempera- 
ture, are important parameters. In the case of phage libraries, note that phage dilution 
buffer already contains Mg + . In some cases, 10% of DMSO in the PCR buffer may help to 
produce a clean, specific PCR product. For a small fragment, it may be necessary to 
amplify more than 30 cycles ( 7). Careful precautions to avoid contamination are essential 
because of the extremely high sensitivity of PCR (11). 

5. Here, an example of this rapid cDNA cloning by PCR screening (RC-PCR) from a bacte- 
ria library is illustrated. In an attempt to clone gene family members of transforming 
growth factor-f! (TGF-f!) receptors, a novel clone, termed clone Bl, was isolated from 
GH 3 rat pituitary tumor cells by PCR using degenerate oligonucleotide primers (4,12). 
The method of RC-PCR was used to isolate a full-length B 1 cDN A from the GH3 cell 
library (12). 

First, a bacteria library of GH3 cells was divided into 16 pools, each of which included 
approx 100,000 clones. As primers for PCR, two oligonucleotides that were expected to 
be highly specific for the B 1 PCR product and that were derived from the sequence of the 
reverse transcription-polymerase chain reaction (RT-PCR) product were utilized: ATC- 
GTGGTTCCGGGAGGCAGAGATC (25-mer, the sequence corresponding to nucleotides 
726-750 of Bl, see Fig. 1A in ref. 12) as a 5'-primer and CTGATTTGGAGCAATGT- 
CTATGGTG (25-mer, nucleotides 1095-1 1 19 of Bl, see Fig. 1A in ref. 12) as a 3'-primer. 
These primers correspond to a region of the kinase domain that is poorly conserved among 
members of the TGF-p" receptor family. 

The screening by PCR was done as follows: After a 5-min denaturation step at 94°C, 
the cycling parameters were 94°C for 45 s, 65°C for 1 min, and 72°C for 2 min, for a total 
of 25 cycles. Three pools of clones generated a PCR product of the expected size. One 
pool generated a slightly larger PCR product; subsequently this fraction was shown to 
contain a B 1 clone with an insertion of 34 nucleotides in the kinase domain. 

For the second screening, this pool of cells was diluted in LB broth to a concentration 
of about 30,000 clones per mL and 100 (iL aliquots of this suspension (about 3000 clones 
per aliquot) were distributed into 96-wells of a microplate. Ten microliters from each 
well in a column were combined (giving a total of 120 (xL from each of the plate's 12 
columns); similarly 10 (xL from each well in a row were combined (giving 80 uL from 
each of the 8 rows) (see Fig. 1). From each of the resulting 20 mixtures, 2.5 \iL were used 
directly in a PCR, and analyzed by gel electrophoresis. Fig. 2 shows the results of the 
second screening; the PCR products were analyzed directly by electrophoresis through 
2% agarose gel and ethidium bromide staini